Stable heterodimeric antibody design with mutations in the Fc domain

ABSTRACT

The provided scaffolds have heavy chains that are asymmetric in the various domains (e.g. CH2 and CH3) to accomplish selectivity between the various Fc receptors involved in modulating effector function, beyond those achievable with a natural homodimeric (symmetric) Fc molecule, and increased stability and purity of the resulting variant Fc heterodimers. These novel molecules comprise complexes of heterogeneous components designed to alter the natural way antibodies behave and that find use in therapeutics.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 61/410,746, filed Nov. 5, 2010; U.S.Provisional Patent Application No. 61/425,375, filed Dec. 21, 2010; U.S.Provisional Patent Application No. 61/439,341, filed Feb. 3, 2011; U.S.Provisional Patent Application No. 61/475,614, filed Apr. 14, 2011; U.S.Provisional Patent Application No. 61/491,846, filed May 31, 2011 andU.S. Provisional Patent Application No. 61/497,861, filed Jun. 16, 2011,each of which is herein incorporated by reference in its entirety.

INTRODUCTION Statement Regarding Sequence Listing

This application incorporates by reference a Sequence Listing submittedwith this application as text file Zymeworks V84467WO.txt created onNov. 4, 2011 and having a size of 15 kilobytes.

FIELD OF THE INVENTION

The present disclosure generally provides polypeptide heterodimers,compositions thereof, and methods for making and using such polypeptideheterodimers. More specifically, the present invention relates tothermo-stable multispecific, including bispecific, antibodies comprisinga heterodimeric Fc domain.

BACKGROUND OF THE INVENTION

Bispecific antibodies are antibody-based molecules that cansimultaneously bind two separate and distinct antigens (or differentepitopes of the same antigen). One use of bispecific antibodies has beento redirect cytotoxic immune effector cells for enhanced killing oftumor cells, such as by antibody dependent cellular cytotoxicity (ADCC).In this context, one arm of the bispecific antibody binds an antigen onthe tumor cell, and the other binds a determinant expressed on effectorcells. By cross-linking tumor and effector cells, the bispecificantibody not only brings the effector cells within the proximity of thetumor cells but also simultaneously triggers their activation, leadingto effective tumor cell-killing. Bispecific antibodies have also beenused to enrich chemo- or radiotherapeutic agents in tumor tissues tominimize detrimental effects to normal tissue. In this setting, one armof the bispecific antibody binds an antigen expressed on the celltargeted for destruction, and the other arm delivers a chemotherapeuticdrug, radioisotope, or toxin.

A major obstacle in the general development of bispecific antibodies hasbeen the difficulty of producing materials of sufficient quality andquantity for both preclinical and clinical studies.

Traditional production of full-length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,1983, Nature, 305:537-539). The intrinsic tendency of the Fc portion ofthe antibody molecule to dimerize leads to the formation of complexmixtures of up to 10 different IgG molecules consisting of variouscombinations of heavy and light chains, of which only one has thecorrect bispecific structure. Purification of the correct molecule,which is usually done by affinity chromatography steps, is rathercumbersome, and the product yields are low. Similar procedures aredisclosed in WO 93/08829, and in Traunecker et al., 1991, EMBO J.,10:3655-3659. Thus, the production of a bispecific antibody moleculewith the two Fab arms selected to bind two different targets usingtraditional hybridoma techniques is challenging [Segal D M et al. (2001)J Immunol Methods. 248, 1-6.]. The trifunctional antibody, Catumaxomab,is a rat/mouse quadroma derived bispecific mAb and the purification ofthis molecule is achieved on the basis of a pH dependent elution inprotein A column based chromatographic separation [Lindhofer H. et al.(1995) J Immunol 155, 219-225].

Another traditional method for bispecific antibody production ischemical conjugation of two antibodies or their fragments havingdifferent specificities. However, this method is also complicated, andthe chemical modification process may inactivate the antibody or promoteaggregation. Because purification from undesired products remainsdifficult, the resulting low yield and poor quality of bispecificantibody make this process unsuitable for the large scale productionrequired for clinical development. In addition, these molecules may notmaintain the traditional antibody conformation.

Recently, various heterodimerization techniques have been used toimprove the production of bispecific antibodies. However, fusion ofsimple heterodimerization domains like the Jun/Fos coiled-coil to scFvdomains lead to a mixture of homo- and heterodimers and need to beassembled by refolding (de Kruif and Logtenberg, J. Biol. Chem. 271:7630-4, 1996). Fusion of scFv fragments to whole antibodies was alsoused as a dimerization device (Coloma and Morrison, Nat. Biotechnol. 15:159-63, 1997). However, such fusion results in a large molecule withpoor solid tissue penetration capabilities. Fusion of two scFv fragmentstogether has also been used to generate bispecific proteins (e.g., BITE®antibodies by Micromet Inc., Bethesda, Md., U.S. Pat. No. 7,635,472).However, such proteins do not contain Fc regions, and thus do not allowmanipulation of their activities via Fc regions. In addition, theseproteins are small (˜55 kDa) and thus have a relatively short half-livein serum.

In other heterodimerization techniques, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., 1986, Methods in Enzymology, 121:210.

According to another approach described in WO96/27011, a pair ofantibody molecules can be engineered to maximize the percentage ofheterodimers that are recovered from recombinant cell culture. In thismethod, one or more small amino acid side chains from the CH3 interfaceof the first antibody molecule are replaced with larger side chains(e.g. tyrosine or tryptophan). Compensatory “cavities” of identical orsimilar size to the large side chain(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chainswith smaller ones (e.g. alanine or threonine). This provides a mechanismfor increasing the yield of the heterodimer over other unwantedend-products such as homodimers [US005731168A, US007183076B2, Ridgway JB, Presta L G, Carter P. Protein Eng 1996 July; 9(7): 617-21; Atwell S,Ridgway J B, Wells J A, Carter P. J Mol Biol 1997 Jul. 4; 270(1):26-35.]. Gunasekaran and coworkers [Gunasekaran K. et al. (2010) J Biol.Chem. 285, 19637-46] have recently employed a complementaryelectrostatic design strategy to achieve the selectiveheterodimerization goal. Davis and coworkers [Davis, J H. et al. (2010)Prot Eng Des Sel; 23(4):195-202] have designed CH3 domains using astrand exchange engineered domain (SEED), which comprises of alternatingsegments of human IgA and IgG CH3 sequences, and these preferentiallyassociate in the form of heterodimers. However, all of thesetechnologies result in antibodies comprising heterodimer Fc regions thatare significantly less stable than the parent or wildtype molecule.

Therefore, there remains a need in the art for alternative multispecificvariant Fc heterodimers, specifically variant CH3 domains, which havebeen modified to select for heterodimers with an increased stability andpurity.

SUMMARY OF THE INVENTION

There is provided according to one aspect of the invention an isolatedheteromultimer comprising a heterodimer Fc region, wherein theheterodimer Fc region comprises a variant CH3 domain comprising aminoacid mutations to promote heterodimer formation, wherein the heterodimerFc region further comprises a variant CH2 domain comprising asymmetricamino acid modifications to promote selective binding of a Fcgammareceptor. In one embodiment the variant CH2 domain selectively bindsFcgamma IIIa receptor as compared to wild-type CH2 domain. In oneembodiment, the variant CH3 domain has a melting temperature (Tm) ofabout 70° C. or greater

There is provided in another aspect an isolated heteromultimercomprising a heterodimer Fc region, wherein the heterodimer Fc regioncomprises a variant CH3 domain comprising amino acid mutations topromote heterodimer formation with increased stability, wherein thevariant CH3 domain has a melting temperature (Tm) of about 70° C. orgreater. In one embodiment, heterodimer Fc region does not comprise anadditional disulfide bond in the CH3 domain relative to a wild type Fcregion, more specifically the heterodimer Fc region does not comprise anadditional disulfide bond in the CH3 domain relative to a wild type Fcregion. In an alternative embodiment, the heterodimer Fc regioncomprises an additional disulfide bond in the variant CH3 domainrelative to a wild type Fc region, with the proviso that the meltingtemperature (Tm) of about 70° C. or greater is in the absence of theadditional disulfide bond. In yet another embodiment, the heterodimer Fcregion comprises an additional disulfide bond in the variant CH3 domainrelative to a wild type Fc region, and wherein the variant CH3 domainhas a melting temperature (Tm) of about 77.5° C. or greater.

Provided in one embodiment, an isolated heteromultimer comprising aheterodimer Fc region, wherein the heterodimer Fc region comprises avariant CH3 domain comprising amino acid mutations to promoteheterodimer formation with increased stability, wherein the variant CH3domain has a melting temperature (Tm) of about 70° C. or greater and theheterodimer Fc region has a purity greater than about 90%, or theheterodimer Fc region has a purity of about 95% or greater or theheterodimer Fc region has a purity of about 98% or greater.

Also provided in one embodiment is an isolated heteromultimer comprisinga heterodimer Fc region, wherein the heterodimer Fc region comprises avariant CH3 domain comprising amino acid mutations to promoteheterodimer formation with increased stability, wherein the variant CH3domain has a melting temperature (Tm) of about 70° C. or greater or theTm is about 71° C. or greater or the Tm is about 74° C. or greater. Inanother embodiment, the heterodimer Fc region has a purity of about 98%or greater and the Tm is about 73° C. or wherein the heterodimer Fcregion has a purity of about 90% or greater and the Tm is about 75° C.

Provided in certain embodiments is an isolated heteromultimer comprisinga heterodimer Fc region, wherein the heterodimer Fc region comprises afirst CH3 domain polypeptide comprising amino acid modifications L351Yand Y407A and a second CH3 domain polypeptide comprising amino acidmodifications T366A and K409F. In one aspect, the first CH3 domainpolypeptide or the second CH3 domain polypeptide comprises a furtheramino acid modification at position T411, D399, S400, F405, N390, orK392. The amino acid modification at position T411 is selected fromT411N, T411R, T411Q, T411K, T411D, T411E or T411W. The amino acidmodification at position D399 is selected from D399R, D399W, D399Y orD399K. The amino acid modification at position S400 is selected fromS400E, S400D, S400R, or S400K. The amino acid modification at positionF405 is selected from F405I, F405M, F405T, F405S, F405V or F405W. Theamino acid modification at position N390 is selected from N390R, N390Kor N390D. The amino acid modification at position K392 is selected fromK392V, K392M, K392R, K392L, K392F or K392E.

In certain embodiments is provided an isolated heteromultimer comprisinga heterodimer Fc region, wherein the heterodimer Fc region comprises afirst CH3 domain polypeptide comprising amino acid modifications L351Yand Y407A and a second CH3 domain polypeptide comprising amino acidmodifications T366V and K409F.

In another embodiment is provided an isolated heteromultimer comprisinga heterodimer Fc region, wherein the heterodimer Fc region comprises afirst CH3 domain polypeptide comprising amino acid modification Y407Aand a second CH3 domain polypeptide comprising amino acid modificationsT366A and K409F. In one aspect the first CH3 domain polypeptide or thesecond CH3 domain polypeptide comprises further amino acid modificationsK392E, T411E, D399R and S400R. In another aspect, the first CH3 domainpolypeptide comprises amino acid modification D399R, S400R and Y407A andthe second CH3 domain polypeptide comprises amino acid modificationT366A, K409F, K392E and T411E. In a further embodiment the variant CH3domain has a melting temperature (Tm) of about 74° C. or greater and theheterodimer has a purity of about 95% or greater.

Provided in another embodiment is an isolated heteromultimer comprisinga heterodimer Fc region, wherein the heterodimer Fc region comprises afirst CH3 domain polypeptide comprising an amino acid modification atpositions L351 and amino acid modification Y407A and a second CH3 domainpolypeptide comprises an amino acid modification at position T366 andamino acid modification K409F. In one aspect the amino acid modificationat position L351 is selected from L351Y, L351I, L351D, L351R or L351F.In another aspect, the amino acid modification at position Y407 isselected from Y407A, Y407V or Y407S. In yet another aspect the aminoacid modification at position T366 is selected from T366A, T366I, T366L,T366M, T366Y, T366S, T366C, T366V or T366W. In one embodiment thevariant CH3 domain has a melting temperature (Tm) of about 75° C. orgreater and the heterodimer has a purity of about 90% or greater.

Provided in another embodiment is an isolated heteromultimer comprisinga heterodimer Fc region, wherein the heterodimer Fc region comprises afirst CH3 domain polypeptide comprising an amino acid modification atposition F405 and amino acid modifications L351Y and Y407V and a secondCH3 domain polypeptide comprises amino acid modification T394W. In oneaspect the first CH3 domain polypeptide or the second CH3 domainpolypeptide comprise an amino acid modification at positions K392, T411,T366, L368 or S400. The amino acid modification at position F405 isF405A, F405I, F405M, F405T, F405S, F405V or F405W. The amino acidmodification at position K392 is K392V, K392M, K392R, K392L, K392F orK392E. The amino acid modification at position T411 is T411N, T411R,T411Q, T411K, T411D, T411E or T411W. The amino acid modification atposition S400 is S400E, S400D, S400R or S400K. The amino acidmodification at position T366 is T366A, T366I, T366L, T366M, T366Y,T366S, T366C, T366V or T366W. The amino acid modification at positionL368 is L368D, L368R, L368T, L368M, L368V, L368F, L368S and L368A.

In another embodiment is provided an isolated heteromultimer comprisinga heterodimer Fc region, wherein the heterodimer Fc region comprises afirst CH3 domain polypeptide comprising an amino acid modificationsL351Y, F405A and Y407V and a second CH3 domain polypeptide comprisesamino acid modification T394W. In one aspect, the second CH3 domainpolypeptide comprises amino acid modification T366L or T366L.

In yet another embodiment is provided an isolated heteromultimercomprising a heterodimer Fc region, wherein the heterodimer Fc regioncomprises a first CH3 domain polypeptide comprising an amino acidmodifications F405A and Y407V and a second CH3 domain polypeptidecomprises amino acid modifications T366I, K392M and T394W.

In certain embodiments are provided an isolated heteromultimercomprising a heterodimer Fc region, wherein the heterodimer Fc regioncomprises a first CH3 domain polypeptide comprising an amino acidmodifications F405A and Y407V and a second CH3 domain polypeptidecomprises amino acid modifications T366L, K392M and T394W.

In another embodiment is provided an isolated heteromultimer comprisinga heterodimer Fc region, wherein the heterodimer Fc region comprises afirst CH3 domain polypeptide comprising an amino acid modificationsF405A and Y407V and a second CH3 domain polypeptide comprises amino acidmodifications T366L and T394W.

In another embodiment is provided an isolated heteromultimer comprisinga heterodimer Fc region, wherein the heterodimer Fc region comprises afirst CH3 domain polypeptide comprising an amino acid modificationsF405A and Y407V and a second CH3 domain polypeptide comprises amino acidmodifications T366I and T394W. In certain embodiments of theheteromultimer is provided bispecific antibody or a multispecificantibody.

In another embodiment is provided a composition comprising aheteromultimer of the invention and a pharmaceutically acceptablecarrier.

In another embodiment is provided a host cell comprising nucleic acidencoding the heteromultimer of the invention.

In certain embodiments is provided heteromultimer, wherein theheteromultimer comprises at least one therapeutic antibody. In oneaspect the therapeutic antibody is selected from the group consisting ofabagovomab, adalimumab, alemtuzumab, aurograb, bapineuzumab,basiliximab, belimumab, bevacizumab, briakinumab, canakinumab,catumaxomab, certolizumab pegol, cetuximab, daclizumab, denosumab,efalizumab, galiximab, gemtuzumab ozogamicin, golimumab, ibritumomabtiuxetan, infliximab, ipilimumab, lumiliximab, mepolizumab, motavizumab,muromonab, mycograb, natalizumab, nimotuzumab, ocrelizumab, ofatumumab,omalizumab, palivizumab, panitumumab, pertuzumab, ranibizumab,reslizumab, rituximab, teplizumab, tocilizumab/atlizumab, tositumomab,trastuzumab, Proxinium™, Rencarex™, ustekinumab, and zalutumumab.

In another embodiment of the heteromultimer of the invention is provideda method of treating cancer in a patient having a cancer characterizedby a cancer antigen, said method comprising administering to saidpatient a therapeutically effective amount of a heteromultimer.

In another embodiment of the heteromultimer of the invention is provideda method of treating immune disorders in a patient having an immunedisorder characterized by an immune antigen, said method comprisingadministering to said patient a therapeutically effective amount of aheteromultimer.

In yet another embodiment is provided an isolated heteromultimercomprising a heterodimer Fc region, wherein the heterodimer Fc regioncomprises a variant CH3 domain comprising amino acid mutations topromote heterodimer formation with increased stability and wherein thevariant CH3 domains are selected from the variants listed in Table 1,Table 6 or Table 7.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical 3-D structure of a wild type antibody showing theCH3 (top), CH2 (middle) and receptor regions. The dotted line rectangleon the left hand side is expanded to the right hand side showing tworegions, Region 1 and Region 2, of the target area of CH3;

FIG. 2 is a graphical 3-D representation of showing the wild typeresidue at position 368;

FIG. 3 is a graphical 3-D representation of Region 1 showing mutatedposition 368;

FIG. 4 is a graphical 3-D representation of additional mutations inRegion 2;

FIG. 5 is a table of in silico calculations for clash score, interfacearea difference, packing different, electrostatic energy difference andoverall “affinity score” for the first three variants AZ1, AZ2 and AZ3;

FIG. 6 shows a graphical 3-D image showing variants AZ2 and AZ3, whichare “built onto” variant AZ1;

FIG. 7 show graphical 3-D representations of AZ2 and AZ3 variants;

FIG. 8 shows a table as in FIG. 5 but for AZ1, AZ2 and AZ3 heterodimers,and homodimers. Affinity score is not relevant for homodimers so thereis no score showing for that aspect for the homodimers;

FIG. 9 is a graphical representation of a 3-D representation of wildtype (left) and mutated AZ4 (right);

FIG. 10 is a table as FIG. 5 showing in silico calculations for AZ4heterodimer and homodimers;

FIG. 11 is a graphical representation of CH3 variants AZ5 (left) and AZ6(right);

FIG. 12 is a table as described for FIG. 5 showing in silico data forAZ4, AZ5 and AZ6;

FIG. 13 is a graphical 3-D representation of an antibody on the left,with a drawing of the possibilities of binding characteristics at thereceptor region using a heterodimeric approach;

FIG. 14 is a schematic representation of the IgG molecule;

FIG. 15 shows multiple sequence alignment of Fcγ receptors.Genebank/Uniprot Sequence ID's: FcγRIIA (sp P12318), FcγRIIB (spP31994), FcγRIIC (gi 126116592), FcγRIIIA (sp P08637), FcγRIIIB (sp075015);

FIG. 16 is a schematic of the crystal structure of Fc-FcγRIIIb Complex[PDB ID: 1T83, Radaev & Sun]. A 1:1 complex of the Fc and Fcγ receptoris observed with an asymmetric contact between the two chains of Fc andthe FcγR;

FIG. 17 shows a schematic of alternative multifunctional molecules basedon the asymmetric Fc scaffold: Asymetric Fc Scaffold and AsymetricFc-Monomeric IgG Arm;

FIG. 18 shows a schematic of alternative multifunctional molecules basedon the asymmetric Fc scaffold: Asymmetric Fc-Monospecific IgG arms andAsymmetric Fc—Bispecific IgG Arm's (Common Light Chain);

FIG. 19 shows an illustration of alternative multifunctional moleculesbased on the asymmetric Fc scaffold. Asymmetric Fc-Bispecific IgG Armsand a functional molecule such as toxin;

FIG. 20 illustrates alternative multifunctional molecules based on theasymmetric Fc scaffold: Asymmetric Fc-Single scFv arm and AsymmetricFc-bispecific scFv Arms;

FIG. 21 illustrations of alternative multifunctional molecules based onthe assymetric Fc scaffold: Asymmetric Fc-Trispecific scFv Arms andAsymmetric Fc-tetraspecific scFv arms.

FIG. 22 displays asymmetric design of mutations on one face of the Fcfor better FcγR selectivity introduces a productive side for FcγRinteractions and a non-productive face with wild type like interactions.Mutations on the non-productive face of the Fc can be introduced toblock interactions with FcR and bias polarity of the Fc so as tointeract on the productive face only.

FIG. 23 shows the amino acid sequence for wild-type human IgG1.

FIG. 24 Shows the iterative process of the Fc heterodimer design,combining positive and negative design strategies as described in detailbelow.

FIG. 25 Shows the in vitro assay used to determine heterodimer purity.The assay is based on a full length monospecific antibody scaffold withtwo Fc heavy chains of different molecular weight; Heavy chain A has aC-terminal HisTag (His) and heavy chain B a C-terminal, cleavable mRFPTag (RFP). The two heavy chains A (His) and B (RFP) are expressed indifferent relative ratios together with a fixed amount of light chain,giving rise to 3 possible dimer species with different molecular weight:a) Homodimer Chain A(His)/Chain A (His) (˜150 kDa); b) Heterodimer ChainA (His)/Chain B (RFP) (˜175 kDa); c) Homodimer Chain B (REP)/Chain B(RFP) (˜200 kDa). After expression, as described in Example 2, the ratioof heterodimer vs. the two homodimers was determined by non-reducingSDS-PAGE, which allows separation of the 3 dimer species by molecularweight. SDS-PAGE gels were stained with Coomassie Brilliant Blue.

FIG. 25A, The variants tested were WT Chain A (His) only; WT chain B(RFP) only; WT chain A (His) plus chain B (RFP); Control 1 chain A (His)plus chain B (RFP), which has a reported heterodimer purity of >95%. Thecomposition of the dimer bands was verified by Western Blot withantibodies directed against the IgG-Fc (anti-Fc), the mRFP Tag(anti-mRFP) and the HisTag (anti-His), as illustrated above. TheSDS-PAGE shows a single band for the His/His hornodimer, a double bandfor the His/RFP heterodimer and multiple bands for the RFP homodimer.The multiple bands are an artifact of the mRFP Tag and have beenconfirmed not to influence the physical properties of the Fcheterodimer.

FIG. 25B. The SDS-PAGE assay was validated with the published Fcheterodimer variants Controls 1-4 as controls, See, Table A. Thevariants were expressed with different relative ratios of chain A (His)vs chain B (RFP): Specifically, Ratio 1:3 is equivalent to a LC, HC_His,HC_mRFP ratio of 25%, 10%, 65%; Ratio 1:1 of 25%, 20%, 55% and Ratio 3:1of 25%, 40%, 35% respectively (the apparent 1:1 expression of chain A(His) to chain B (RFP) has been determined to be close to 20%/55%(His/RFP) for WT Fc).

FIG. 25C. Shows a non-reducing SDS-PAGE assay to determine heterodimerpurity of Scaffold 1 variants. The Fc variants were expressed withdifferent relative ratios of chain A (His) vs chain B (RFP) and analyzedby non-reducing SDS-PAGE as described in FIG. 2. Specifically, Ratio 1:3is equivalent to a LC, HC_His, HC_mRFP ratio of 25%, 10%, 65%; Ratio 1:1of 25%, 20%, 55% and Ratio 3:1 of 25%, 40%, 35% respectively (theapparent 1:1 expression of chain A (His) to chain B (RFP) has beendetermined to be close to 20%155% (His/RFP) for WT Fc).

FIG. 26 Shows Fc Heterodimer variants expressed with a specific ratio ofchain A (His) vs chain B (RFP) (See, Table 2), purified by Protein Aaffinity chromatography and analyzed by non-reducing SDS-PAGE asdescribed in FIG. 25.

FIG. 26A Illustrates how different variants have been binned intocategories of purity based on the visual inspection of the SDS-PAGEresults. For comparison the equivalent amount of Protein A purifiedproduct was loaded on the gel. This definition of purity based onnon-reducing SDS-PAGE has been confirmed by LC/MS on selected variants(see FIG. 28).

FIG. 26B Example SDS-PAGE results of selected Protein A purifiedheterodimer variants of Scaffold 1 and 2 (AZ94, AZ86, AZ70, AZ33 andAZ34).

FIG. 27 Illustrates DSC analysis to determine the melting temperature ofthe CH3-CH3 domain wherein two independent methods were used.

FIG. 27A. The thermograms were fitted to 4 independentNon-2-State-transitions and optimized to yield values for the CH2 andFab transitions close to the reported literature values for Herceptin of˜72° C. (CH2) and ˜82° C. (Fab).

FIG. 27B. The normalized and baseline corrected thermograms for theheterodimer variants were subtracted from the WT to yield a positive andnegative difference peak for only the CH3 transition.

FIG. 28 Illustrates the LC/MS analysis of example variant AZ70 asdescribed in the example 2. The expected (calculated average) masses forthe glycosylated heterodimer and homodimers are indicated. The regionconsistent with the heterodimer mass contains major peaks correspondingto the loss of a glycine (−57 Da) and the addition of 1 or 2 hexoses(+162 Da and +324 Da, respectively). The Heterodimer purity isclassified as >90% if there are no significant peaks corresponding toeither of the homodimers.

FIG. 29 Shows the CH3 interface of FIG. 29A WT Fc; FIG. 29B AZ6; FIG.29C AZ33; FIG. 29D AZ19. The comprehensive in silico analysis, asdescribed in the detailed description section, and the comparison of thevariants to the WT indicated that one of the reasons for the lower thanWT stability of the initial AZ33 heterodimer is the loss of the coreinteraction/packing of Y407 and T366. The initial AZ33 shows non-optimalpacking at this hydrophobic core as illustrated FIG. 29B, suggestingthat optimization of this region, particularly at position T366 wouldimprove the stability of AZ33. This is illustrated in FIG. 29C and FIG.29D with T366I and T366L. The experimental data correlates with thisstructural analysis and shows that T366L gives the greatest improvementin Tm. See, Example 5.

FIG. 30 Illustrates the utility and importance of the conformationaldynamics analysis, exemplified at the initial Scaffold 1 variant AZ8.The structure after in silico mutagenesis (backbone conformation closeto WT) is superimposed with a representative structure of a 50 nsMolecular Dynamics simulation analysis. The figure highlights the largeconformational difference in the loop region D399-S400 of AZ8 variantvs. WT, which in turn exposes the hydrophobic core to solvent and causesdecreased stability of the AZ8 heterodimer.

FIG. 31 illustrates how the information from the comprehensive in silicoanalysis and the MD simulation was used in the described positive designstrategy. As illustrated in FIG. 30, one of the reasons for the lowerthan WT stability of AZ8 is the weakened interaction of the loop 399-400to 409, which is mainly due to the loss of the F405 packing interactions(see comparison of FIG. 31A (WT) vs FIG. 31B (AZ8)). One of the positivedesign strategies was optimization of the hydrophobic packing of area,to stabilize the 399-400 loop conformation. This was achieved by theK392M mutation that is illustrated in FIG. 31C. FIG. 31C represents theheterodimer AZ33, which has a Tm of 74° vs. 68° of the initial negativedesign variant AZ8.

FIG. 32 Illustrates the dynamics of the Fc molecule observed usingprincipal component analysis of a molecular dynamics trajectory. FIG.32A shows a backbone trace of the Fc structure as reference. FIGS. 32Band C represent an overlay of dynamics observed along the top 2principal modes of motion in the Fc structure. The CH2 domains of chainA and B exhibits significant opening/closing motion relative to eachother while the CH3 domains are relatively rigid. Mutations at the CH3interface impact the relative flexibility and dynamics of thisopen/close motion in the CH2 domains.

FIG. 33 Illustrates the hydrophobic core packing of two Scaffold-2variants vs. WT. FIG. 33A WT Fc; FIG. 33B AZ63; and FIG. 33C AZ70. Thecomprehensive in-silico analysis of the initial Scaffold-2 variantsuggested that loss of the core WT interactions of Y407-T366 is one ofthe reasons for the lower than WT stability for the initial Scaffold-2variants. The loss of Y407-T366 is partially compensated by themutations K409F, but as illustrated in FIG. 33B, particularly the T366Amutation leaves a cavity in the hydrophobic core, which destabilizes thevariant vs. WT. Targeting this hydrophobic core by additional mutationsT366V_L351Y, as shown by Fc variant AZ70 in FIG. 33C, proved to besuccessful; AZ70 has an experimentally determined Tm of 75.5° C. See,Table 4 and Example 6.

FIG. 34 Illustrates the interactions of the loop 399-400 of twoScaffold-2 variants vs. the WT: FIG. 34A WT Fc; FIG. 34B AZ63; and FIG.34C AZ94. The comprehensive in-silico analysis of the initial Scaffold-2variant suggested that loss of the WT salt-bridge K409-D399 (FIG. 34A)due to the mutation K409F and the hence unsatisfied D399 (FIG. 34B)causes a more ‘open’ conformation of the 399-400 loop. This leadsfurthermore to a greater solvent exposure of the hydrophobic core and afurther destabilization of the variant vs WT. One of the strategiesemployed to stabilize the 399-400 loop and compensate for the loss ofthe K409-D399 interaction was the design of additional salt bridgesD399R-T411E and S400R-K392E as illustrated in FIG. 34C for variant AZ94.Experimental data showed a purity of >95% and Tm of 74° C. See, Table 4and Example 6. Further, although AZ94 has a considerably higher purityand stability compared to the initial Scaffold-2 variant (purity <90%,Tm 71° C.), the hydrophobic core mutations of AZ94 are less preferredthan the ‘best’ hydrophobic core mutations identified in variant AZ70(FIG. 33). Since the mutations at the hydrophobic core in AZ70(T366V_L351Y) are distal from the salt-bridge mutations of AZ94 at theloop 399-400, the combination of AZ70 amino acid mutations and theadditional AZ94 mutations, is expected to have a higher meltingtemperature then AZ70 or AZ94. This combination can be tested asdescribed in Examples 1-4.

FIG. 35 Illustrates the association constant (Ka(M⁻¹)) of homodimericIgG1 Fc, the heterodimeric variants het1(Control 1):A:Y349C_T366S_L368A_Y407V/B:S354C_T366W and het2(Control 4):A:K409D_K392D/B:D399K_D356K binding to the six Fcgamma receptors. Theheterodimeric Fc variants tend to show slightly altered binding to theFcgamma receptors compared to the wild type IgG1 Fc. See, Example 7

FIG. 36A Shows the relative binding strength of a wild type IgG1 Fc andits various homodimeric and asymmetric mutant forms to the IIbF, IIBYand IIaR receptors, based on the wild type binding strength asreference. (Homo Fc+S267D) refers to the binding strength of ahomodimeric Fc with the S267D mutation on both chains. (Het Fc+asymS267D) refers to the binding strength of a heterodimeric Fc with theS267D mutation introduced in one of the two chains in Fc. The average ofthe binding strength obtained by introducing the mutation on either ofthe two Fc chains is reported. Introduction of this mutation on onechain reduced the binding strength to roughly half the strength observedfor the same mutation in a homodimeric manner. The (Het Fc+asymS267D+asym E269K) refers to the binding strength of a heterodimeric Fcwith both the S267D and E269K mutations introduced in an asymmetricmanner on one of the two Fc chains. The E269K mutation blocks theinteraction of the FcgR to one of the faces of the Fc and is able tobring down the binding strength by roughly half of what was observed forthe asymmetric S267D variant (Het Fc+S267D) by itself. The Het Fc hereis comprised of CH3 mutations as indicated for the variant het2 (Control4) in FIG. 35.

FIG. 36B Shows the association constant (Ka(M⁻¹)) of various Fc's andits variants with a number of FcgRIIa, FcgRIIb and FcgRIIIa allotypes.The Ka of wild type IgG1 Fc to various Fcg receptors is represented ascolumns with horizontal shade. The bars with vertical shades (homodimerbase2) represent the Ka of homodimeric Fc with the mutationsS239D/D265S/I332E/S298A. The columns with the slanted shade representthe Ka of heterodimeric Fc with asymmetric mutationsA:S239D/D265S/I332E/E269K and B:S239D/D265S/S298A in the CH2 domain. Theintroduction of asymmetric mutations is able to achieve increasedselectivity between the IIIa and IIa/IIb receptors. The Heterodimeric Fchere is comprised of CH3 mutations as indicated for the variant het2(Control 4) in FIG. 35.

FIG. 36C Shows the association constant (Ka (M⁻¹)) for wild type IgG1and three other variants involving homodimeric or asymmetric mutationsin the CH2 domain of the Fc region. The Ka of wild type Fc isrepresented in the column shaded with grids. The Ka of Fc variant withthe base mutation S239D/K326E/A330L/I332E/S298A introduced in ahomodimeric manner (homodimer base1) on both the chains of Fc is shownwith the slanted patterned column. Introduction of related mutations inan asymmetric manner in chains A and B of a heterodimeric Fc (heterobase1) is shown with the horizontal lines. The column with verticalshaded lines represents the asymmetric variant including the E269Kmutation (hetero base 1+PD). The Heterodimeric Fc here is comprised ofCH3 mutations as indicated for the variant het2 (Control 4) in FIG. 35.

FIG. 37—Table 6 Is a list of variants CH3 domains based on the thirddesign phase as described in Example 5 for Scaffold 1.

FIG. 38—Table 7 is a list of variant CH3 domains based on the thirddesign phase as described in Example 6 for scaffold 2.

DETAILED DESCRIPTION

The present invention provides modified CH3 domains comprising specificamino acid modifications to promote heterodimer formation. In oneembodiment, the modified CH3 domains comprise specific amino acidmodifications to promote heterodimer formation (See, for example Table1). In another embodiment the modified CH3 domains comprise specificamino acid modifications to promote heterodimer formation with increasedstability (See, for example Table 4, Table 6 and Table 7). Stability ismeasured as the melting temperature (Tm) of the CH3 domain and anincreased stability refers to a Tm of about 70° C. or greater. The CH3domains form part of the Fc region of a heteromultimeric or bispecificantibody. Thus, provided herein in one embodiment are heteromultimerscomprising a heterodimer Fc region, wherein the heterodimer Fc regioncomprises a modified or variant CH3 domain comprising amino acidmutations to promote heterodimer formation wherein the variant CH3domains are selected from the variants listed in Table 1. In a secondembodiment, provided are heteromultimers comprising a heterodimer Fcregion, wherein the heterodimer Fc region comprises a variant CH3 domaincomprising amino acid mutations to promote heterodimer formation withincreased stability, wherein the variant CH3 domain has a meltingtemperature (Tm) of about 70° C. or greater.

Amino acid modifications that may be utilized to generate a modified CH3domain include, but are not limited to, amino acid insertions,deletions, substitutions, and rearrangements. The modifications of theCH3 domain and the modified CH3 domains are referred to hereincollectively as “CH3 modifications”, “modified CH3 domains”, “variantCH3 domains” or “CH3 variants”. These modified CH3 domains may beincorporated into a molecule of choice. Accordingly, in one embodimentare provided molecules, in particular polypeptides, more specificallyimmunoglobulins (e.g., antibodies) and other binding proteins,comprising an Fc region (as used herein “Fc region” and similar termsencompass any heavy chain constant region domain comprising at least aportion of the CH3 domain) incorporating a modified CH3 domain.Molecules comprising Fc regions comprising a modified CH3 domain (e.g.,a CH3 domain comprising one or more amino acid insertions, deletions,substitutions, or rearrangements) are referred to herein as “Fcvariants”, “heterodimers” or “heteromultimers”. The present Fc variantscomprise a CH3 domain that has been asymmetrically modified to generateheterodimer Fc variants or regions. The Fc region is comprised of twoheavy chain constant domain polypetides—Chain A and Chain B, which canbe used interchangeably provided that each Fc region comprises one ChainA and one Chain B polypeptide. The amino acid modifications areintroduced into the CH3 in an asymmetric fashion resulting in aheterodimer when two modified CH3 domains form an Fc variant (See, e.g.,Table 1). As used herein, asymmetric amino acid modifications are anymodification wherein an amino acid at a specific position on onepolypeptide (e.g., “Chain A”) is different from the amino acid on thesecond polypeptide (e.g., “Chain B”) at the same position of theheterodimer or Fc variant. This can be a result of modification of onlyone of the two amino acids or modification of both amino acids to twodifferent amino acids from Chain A and Chain B of the Fc variant. It isunderstood that the variant CH3 domains comprise one or more asymmetricamino acid modifications.

In the present description, any concentration range, percentage range,ratio range, or integer range is to be understood to include the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated. As used herein, “about” means±10% of the indicatedrange, value, sequence, or structure, unless otherwise indicated. Itshould be understood that the terms “a” and “an” as used herein refer to“one or more” of the enumerated components unless otherwise indicated ordictated by its context. The use of the alternative (e.g., “or”) shouldbe understood to mean either one, both, or any combination thereof ofthe alternatives. As used herein, the terms “include” and “comprise” areused synonymously. In addition, it should be understood that theindividual single chain polypeptides or heterodimers derived fromvarious combinations of the structures and substituents (e.g., variantCH3 domains) described herein are disclosed by the present applicationto the same extent as if each single chain polypeptide or heterodimerwere set forth individually. Thus, selection of particular components toform individual single chain polypeptides or heterodimers is within thescope of the present disclosure.

The “first polypeptide” is any polypeptide that is to be associated witha second polypeptide, also referred to herein as “Chain A”. The firstand second polypeptide meet at an “interface”. The “second polypeptide”is any polypeptide that is to be associated with the first polypeptidevia an “interface”, also referred to herein as “Chain B”. The“interface” comprises those “contact” amino acid residues in the firstpolypeptide that interact with one or more “contact” amino acid residuesin the interface of the second polypeptide. As used herein, theinterface comprises the CH3 domain of an Fc region that preferably isderived from an IgG antibody and most preferably a human IgG₁ antibody.

As used herein, “isolated” heteromultimer means a heteromultimer thathas been identified and separated and/or recovered from a component ofits natural cell culture environment. Contaminant components of itsnatural environment are materials that would interfere with diagnosticor therapeutic uses for the heteromultimer, and may include enzymes,hormones, and other proteinaceous or non-proteinaceous solutes.

The variant Fc heterodimers are generally purified to substantialhomogeneity. The phrases “substantially homogeneous”, “substantiallyhomogeneous form” and “substantial homogeneity” are used to indicatethat the product is substantially devoid of by-products originated fromundesired polypeptide combinations (e.g. homodimers). Expressed in termsof purity, substantial homogeneity means that the amount of by-productsdoes not exceed 10%, and preferably is below 5%, more preferably below1%, most preferably below 0.5%, wherein the percentages are by weight.

Terms understood by those in the art of antibody technology are eachgiven the meaning acquired in the art, unless expressly defineddifferently herein. Antibodies are known to have variable regions, ahinge region, and constant domains. Immunoglobulin structure andfunction are reviewed, for example, in Harlow et al, Eds., Antibodies: ALaboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, ColdSpring Harbor, 1988).

The design of variant Fc heterodimers from wildtype homodimers isillustrated by the concept of positive and negative design in thecontext of protein engineering by balancing stability vs. specificity,wherein mutations are introduced with the goal of driving heterodimerformation over homodimer formation when the polypeptides are expressedin cell culture conditions. Negative design strategies maximizeunfavorable interactions for the formation of homodimers, by eitherintroducing bulky sidechains on one chain and small sidechains on theopposite, for example the knobs-into-holes strategy developed byGenentech (Ridgway J B, Presta L G, Carter P. ‘Knobs-into-holes’engineering of antibody CH3 domains for heavy chain heterodimerization.Protein Eng. 1996 July; 9(7):617-21; Atwell S, Ridgway J B, Wells J A,Carter P. Stable heterodimers from remodeling the domain interface of ahomodimer using a phage display library. J Mol. Biol. 270(1):26-35(1997))), or by electrostatic engineering that leads to repulsion ofhomodimer formation, for example the electrostatic steering strategydeveloped by Amgen (Gunaskekaran K, et al. Enhancing antibody Fcheterodimer formation through electrostatic steering effects:applications to bispecific molecules and monovalent IgG. JBC 285 (25):19637-19646 (2010)). In these two examples, negative design asymmetricpoint mutations were introduced into the wild-type CH3 domain to driveheterodimer formation. To date, only negative design strategies havebeen used to develop Fc heterodimers. Published results show thatheterodimers designed using only a negative design approach leads tohigh specificity with >95% heterodimers, but destabilizes the complexconsiderably (Supra). These negative design heterodimers posses amelting temperature, of the modified CH3 domain, of 69° C. or less,absent additional disulfide bonds as compared to the wild type. See,Table A below.

TABLE A Published Fc Heterodimer Antibodies. Engi- neering Tm ChainsApproach Source Purity ° C. Wild- — 81-83 Type — Con- Y349C_T366S_(—)Knobs- Genentech 95%  >77** trol 4 L368A_Y407V into- (MerchantS354C_T366W holes plus et al.) disulfide Con- K409D_K392D Electro- Amgen<80%  NP trol 3 D399K static (Gunaskekaran steering et al.) Con-T366S_L368A_(—) Knobs- Genentech 95% 69 trol 2 Y407V into- (Atwell T366Wholes et al.) Con- K409D_K392D Electro- Amgen 100%* 67 trol 1D399K_E356K static (Gunaskekaran steering et al.) Con- IgG-IgA StrandEMD Serono >90%  68 trol 5 chimera Exchange (Muda et al.) *We observed apurity of >90% for Control 1 in our assay system, but not 100% aspreviously reported in the literature. **We observed a Tm greater than77° C. for control 4 in our assay system; the Tm for this variant hasnot been published in the literature. NP - The Tm for Control 3 has notbeen published and it was not tested in our assays systems.

The melting temperature for wild-type IgG1 is shown as a range from81-83 as the values in the literature vary depending on the assay systemused, we report a value of 81.5° C. in our assay system.

In contrast to negative design, a general concept used to engineerproteins is positive design. In this instance amino acid modificationsare introduced into polypeptides to maximize favorable interactionswithin or between proteins. This strategy assumes that when introducingmultiple mutations that specifically stabilize the desired heterodimerwhile neglecting the effect on the homodimers, the net effect will bebetter specificity for the desired heterodimer interactions over thehomodimers and hence a greater heterodimer specificity. It is understoodin the context of protein engineering that positive design strategiesoptimize the stability of the desired protein interactions, but rarelyachieve >90% specificity (Havranek J J & Harbury P B. Automated designof specificity in molecular recognition. Nat Struct Biol. 10(1):45-52(2003); Bolan D N, Grant R A, Baker T A, Sauer R T. Specificity versusstability in computational protein design. Proc Natl Acad Sci USA. 6;102(36):12724-9 (2005); Huang P S, Love J J, Mayo S L. A de novodesigned protein protein interface Protein Sci. 16(12):2770-4 (2007)).And thus, to date, positive design strategies have not been used todesign Fc heterodimers as specificity was more important than stabilityfor therapeutic antibody manufacturing and development. In addition,beneficial positive design mutations can be hard to predict. Othermethodologies for improving stability, such as additional disulfidebonds, have been tried to improve stabilityin Fc heterodimers withlimited success on improvements to the molecule. (See, Table A) This maybe because all engineered Fc CH3 domain disulfide bonds are solventexposed, which results in a short lifetime of the disulfide bond andtherefore a significant impact on the longterm stability of theheterodimer—especially when the engineered CH3 domain has a Tm of lessthan 70° C. without the additional disulfide bond (as in Control 4 whichhas a Tm of 69° C. without the disulfide (see Control 2). It iscontemplated that other methodologies to improve stability, such asdisulfide bonds, can also be used with the present Fc variants, providedthe intrinsic stability (measured as melting temperature) of the CH3domain is 70° C. or greater without the disulfide bond, in particularwhen the intrinsic stability (measured as melting temperature) of theCH3 domain is 72° C. or greater without the disulfide bond.

Therefore, we herein disclose a novel method for designing Fcheterodimers that results in both stable and highly specific heterodimerformation. This design method combines both negative and positive designstrategies along with structural and computational modeling guidedprotein engineering techniques. This powerful method has allowed us todesign novel combinations of mutations in the IgG1 CH3 domain whereinusing only standard cell culture conditions heterodimers were formedwith more than 90% purity compared to homodimers and the resultingheterodimers had a melting temperature of 70° C. or greater. Inexemplary embodiments, the Fc variant heterodimers have a meltingtemperature of 73° C. or greater and a purity of greater than 98%. Inother exemplary embodiments, the Fc variant heterodimers have a meltingtemperature of 75° C. or greater and a purity of greater than 90%.

In certain embodiments, an isolated heteromultimer comprising aheterodimer Fc region is provided wherein the heterodimer Fc regioncomprises a variant CH3 domain comprising amino acid mutations topromote heterodimer formation with increased stability, wherein thevariant CH3 domain has a melting temperature (Tm) of 70° C. or greater.As used herein “increased stability” or “stable heterodimer”, refers toa variant CH3 domain, in heterodimer formation, with a meltingtemperature of about 70° C. or greater. In addition, it is understoodthat the term “to promote heterodimer formation” refers herein to theamino acid mutations in the CH3 domain that result in greater than 90%heterodimer formation compared to homodimer formation.

In a further embodiment, this increased stability is in the absence ofan additional disulfide bond. Specifically, the increased stability isin the absence of an additional disulfide bond in the CH3 domain. In oneembodiment, the variant CH3 domain does not comprise an additionaldisulfide bond as compared to wild-type CH3 domain. In an alternativeembodiment, the variant CH3 comprises at least one disulfide bond ascompared to wild-type CH3 domain, provided that the variant CH3 has amelting temperature of 70° C. or greater in the absence of the disulfidebond. In one embodiments, the variant CH3 domain comprises at least onedisulfide bond as compared to wild-type CH3 domain, and the variant CH3domain has a melting temperature (Tm) of about 77.5° C. or greater. Inan embodiment, the variant CH3 domain comprises at least one disulfidebond as compared to wild-type CH3 domain, and the variant CH3 domain hasa melting temperature (Tm) of about 78° C. or greater. In anotherembodiment, the variant CH3 domain comprises at least one disulfide bondas compared to wild-type CH3 domain, and the variant CH3 domain has amelting temperature (Tm) of greater than about 78° C., or greater thanabout 78.5° C., or greater than about 79° C., or greater than about79.5° C., or greater than about 80° C., or greater than about 80.5° C.,or greater than about 81° C.

In one embodiment, the variant CH3 domain has a melting temperature ofgreater than about 70° C., or greater than about 70.5° C., or greaterthan about 71° C., or greater than about 71.5° C., or greater than about72° C., or greater than about 72.5° C., or greater than about 73° C., orgreater than about 73.5° C., or greater than about 74° C., or greaterthan about 74.5° C., or greater than about 75° C., or greater than about75.5° C., or greater than about 76° C., or greater than about 76.5° C.,or greater than about 77° C., or greater than about 77.5° C., or greaterthan about 78° C., or greater than about 78.5° C., or greater than about79° C., or greater than about 79.5° C., or greater than about 80° C., orgreater than about 80.5° C., or greater than about 81° C. In anotherembodiment, the variant CH3 domain has a melting temperature of about70° C., or about 70.5° C., or about 71° C., or about 71.5° C., or about72° C., or about 72.5° C., or about 73° C., or about 73.5° C., or about74° C., or about 74.5° C., or about 75° C., or about 75.5° C., or about76° C., or about 76.5° C., or about 77° C., or about 77.5° C., or about78° C., or about 78.5° C., or about 79° C., or about 79.5° C., or about80° C., or about 80.5° C., or about 81° C. In yet another embodiment,the variant CH3 domain has a melting temperature of about 70° C. toabout 81° C., or about 70.5° C. to about 81° C., or about 71° C. toabout 81° C., or about 71.5° C. to about 81° C., or about 72° C. toabout 81° C., or about 72.5° C. to about 81° C., or about 73° C. toabout 81° C., or about 73.5° C. to about 81° C., or about 74° C. toabout 81° C., or about 74.5° C. to about 81° C., or about 75° C. toabout 81° C., or about 75.5° C. to about 81° C., or 76° C. to about 81°C., or about 76.5° C. to about 81° C., or about 77° C. to about 81° C.,or about 77.5° C. to about 81° C., or about 78° C. to about 81° C., orabout 78.5° C. to about 81° C., or about 79° C. to about 81° C. In yetanother embodiment, the variant CH3 domain has a melting temperature ofabout 71° C. to about 76° C., or about 72° C. to about 76° C., or about73° C. to about 76° C., or about 74° C. to about 76° C.

In addition to improved stability, the heterodimer Fc region comprises avariant CH3 domain comprising amino acid mutations to promoteheterodimer formation. It is understood that these amino acid mutationsto promote heterodimer formation are as compared to homodimer formation.This heterodimer formation as compared to homodimer formation isreferred jointly herein as “purity” or “specificity” or “heterodimerpurity” or “heterodimer specificity”. It is understood that theheterodimer purity refers to the percentage of desired heterodimerformed as compared to homodimer species formed in solution understandard cell culture conditions prior to selective purification of theheterodimer species. For instance, a heterodimer purity of 90% indicatesthat 90% of the dimer species in solution is the desired heterodimer. Inone embodiment, the Fc variant heterodimers have a purity of greaterthan about 90%, or greater than about 91%, or greater than about 92%, orgreater than about 93%, or greater than about 94%, or greater than about95%, or greater than about 96%, or greater than about 97%, or greaterthan about 98%, or greater than about 99%. In another embodiment, the Fcvariant heterodimers have a purity of about 90%, or about 91%, or about92%, or about 93%, or about 94%, or about 95%, or about 96%, or about97%, or about 98%, or about 99%, or about 100%.

In a specific embodiment, the isolated heteromultimer comprising aheterodimer Fc region, wherein the heterodimer Fc region comprises avariant CH3 domain comprising amino acid mutations to promoteheterodimer formation with increased stability, wherein the variant CH3domain has a melting temperature (Tm) of 70° C. or greater and theresulting heterodimer has a purity greater than 90%. In one aspect, theresulting Fc variant heterodimer has a purity greater than 90% and thevariant CH3 domain has a melting temperature of greater than about 70°C., or greater than about 71° C., or greater than about 72° C., orgreater than about 73° C., or greater than about 74° C., or greater thanabout 75° C., or greater than about 76° C., or greater than about 77°C., or greater than about 78° C., or greater than about 79° C., orgreater than about 80° C. or greater than about 81° C. In a furtheraspect, the variant CH3 domain has a melting temperature of 70° C. orgreater and the resulting Fc variant heterodimer has a purity greaterthan about 90%, or greater than about 91%, or greater than about 92%, orgreater than about 93%, or greater than about 94%, or greater than about95%, or greater than about 96%, or greater than about 97%, or greaterthan about 98%, or greater than about 99%.

In order to design these Fc variants with improved stability and puritywe employed an iterative process of computational design andexperimental screening to select the most successful combinations ofpositive and negative design strategies (See, FIG. 24).

Specifically, in the initial design phase different negative design Fcvariant heterodimers were made and tested for expression and stabilityas described in Examples 1-3. The initial design phase included Fcvariant heterodimers AZ1-AZ16 (See, Table 1). From this initial set ofnegative design Fc variant heterodimers, which were expected to have lowstability (e.g., a Tm of less than 71° C.), the Fc variant heterodimerswith greater than 90% purity and a melting temperature of about 68° C.or greater were selected for further development. This included Fcvariant heterodimers AZ6, AZ8 and AZ15. In the second design phase,those selected Fc variant heterodimers were further modified to driveboth stability and purity using positive design strategies following adetailed computational and structural analysis. The selected Fc variantheterodimers (AZ6, AZ8, and AZ15) were each analyzed with computationalmethods and comprehensive structure function analysis to identify thestructural reasons these Fc variants had a lower stability than thewild-type Fc homodimer, which is 81° C. for IgG1. See, Table 4 for thelist of Fc variant heterodimers and the Tm values.

In certain embodiments, the variant CH3 domain is selected from AZ1, orAZ2, or AZ3, or AZ4, or AZ5, or AZ6, or AZ7, or AZ8, or AZ9, or AZ10, orAZ11, or AZ12, or AZ13, or AZ14, or AZ15, or AZ16. In selectedembodiments, the variant CH3 domain is AZ6, or AZ8 or AZ15.

The computational tools and structure-function analysis included, butwere not limited to molecular dynamic analysis (MD), sidechain/backbonere-packing, Knowledge Base Potential (KBP), cavity and (hydrophobic)packing analysis (LJ, CCSD, SASA, dSASA (carbon/all-atom)),electrostatic-GB calculations, and coupling analysis. (See, FIG. 24 foran overview of the computational strategy)

An aspect of our protein engineering approach relied on combiningstructural information of the Fc IgG protein derived from X-raycrystallography with computational modeling and simulation of the wildtype and variant forms of the CH3 domain. This allowed us to gain novelstructural and physico-chemical insights about the potential role ofindividual amino acids and their cooperative action. These structuraland physico-chemical insights, obtained from multiple variant CH3domains, along with the resulting empirical data pertaining to theirstability and purity helped us develop an understanding for therelationship between purity and stability of the Fc heterodimer ascompared to the Fc homodimers and the simulated structural models. Inorder to execute our simulations we started by building complete andrealistic models and refining the quality of the wild type Fc structureof an IgG1 antibody. Protein structures derived from X-raycrystallography are lacking in detail regarding certain features of theprotein in aqueous medium under physiological condition and ourrefinement procedures addressed these limitations. These includebuilding missing regions of the protein structure, often flexibleportions of the protein such as loops and some residue side chains,evaluating and defining the protonation states of the neutral andcharged residues and placement of potential functionally relevant watermolecules associated with the protein.

Molecular dynamics (MD) algorithms are one tool we used, by simulatingthe protein structure, to evaluate the intrinsic dynamic nature of theFc homodimer and the variant CH3 domains in an aqueous environment.Molecular dynamics simulations track the dynamic trajectory of amolecule resulting from motions arising out of interactions and forcesacting between all the atomic entities in the protein and its localenvironment, in this case the atoms constituting the Fc and itssurrounding water molecules. Following molecular dynamics simulations,various aspects of the trajectories were analyzed to gain insight intothe structural and dynamic characteristics of the Fc homodimer andvariant Fc heterodimer, which we used to identify specific amino acidmutations to improve both purity and stability of the molecule.

Therefore, the generated MD trajectories were studied using methods suchas the principal component analysis to reveal the intrinsic lowfrequency modes of motion in the Fc structure. This provides insightinto the potential conformational sub-states of the protein (See, FIG.32). While the critical protein-protein interactions between chain A andB in the Fc region occur at the interface of the CH3 domains, oursimulations indicated that this interface acts as a hinge in a motionthat involves the “opening” and “closing” of the N-terminal ends of theCH2 domains relative to each other. The CH2 domain interacts with FcgR'sat this end as seen in FIG. 16. Thus, while not wishing to be bound by atheory, it appears that introduction of amino acid mutations at the CH3interface impacts the magnitude and nature of the open/close motion atthe N-terminal end of the Fc and therefore how the Fc interacts with theFcgR's. See, example 4 and Table 5.

The generated MD trajectories were also studied to determine themutability of specific amino acid residue positions in the Fc structurebased on profiling their flexibility and analysis of their environment.This algorithm allowed us to identify residues that could affect proteinstructure and function, providing unique insight into residuecharacteristics and mutability for subsequent design phases of thevariant CH3 domains. This analysis also enabled us to compare multiplesimulations, and assess mutability based on outliers followingprofiling.

The generated MD trajectories were also studied to determine correlatedresidue motions in the protein and the formation of networks of residuesas a result of coupling between them. Finding dynamic correlations andnetworks of residues within the Fc structure is a critical step inunderstanding the protein as a dynamic entity and for developing insightinto the effects of mutations at distal sites. See, e.g. Example 6

Thus, we studied in detail the impact of mutations on the localenvironment of the site of mutation. The formation of a well packed coreat the CH3 interface between chain A and B is critical for thespontaneous pairing of the two chains in a stable Fc structure. Goodpacking is the result of strong structural complementarity betweeninteracting molecular partners coupled with favorable interactionsbetween the contacting groups. The favorable interactions result fromeither buried hydrophobic contacts well removed from solvent exposureand/or from the formation of complementary electrostatic contactsbetween hydrophilic polar groups. These hydrophobic and hydrophiliccontacts have entropic and enthalpic contributions to the free energy ofdimer formation at the CH3 interface. We employ a variety of algorithmsto accurately model the packing at the CH3 interface between chain A andchain B and subsequently evaluate the thermodynamic properties of theinterface by scoring a number of relevant physicochemical properties.

We employed a number of protein packing methods including flexiblebackbones to optimize and prepare model structures for the large numberof variants we computationally screened. Following packing we evaluateda number of terms including contact density, clash score, hydrogenbonds, hydrophobicity and electrostatics. The use of the solvationmodels allowed us to more accurately address the effect of solventenvironment and contrast the free energy differences following mutationof specific positions in the protein to alternate residue types. Contactdensity and clash score provide a measure of complementarity, a criticalaspect of effective protein packing. These screening procedures arebased on the application of knowledge-based potentials or couplinganalysis schemes relying on pair-wise residue interaction energy andentropy computations.

This comprehensive in-silica analysis provided a detailed understandingof the differences of each Fc variant compared to wild-type with respectto interface hotspots, sites of asymmetry, cavities and poorly packedregions, structural dynamics of individual sites and sites of localunfolding. These combined results of the described computationalanalysis identified specific residues, sequence/structural motifs andcavities that were not optimized and in combination responsible for thelower stability (e.g., Tm of 68° C.) and/or lower specificity of <90%purity. In the second design phase we used targeted positive design tospecifically address these hypothesis by additional point-mutations andtested these by in-silica engineering using the above describedmethodology and analysis (See, FIG. 24). The Fc variant heterodimersdesigned to improve stability and purity for each targeted design inphase two (Fc variant heterodimers AZ17-AZ101) were validatedexperimentally for expression and stability as described in Examples1-4.

In certain embodiments, provided herein are isolated heteromultimerscomprising a heterodimer Fc region, wherein the heterodimer Fc regioncomprises a variant CH3 domain comprising amino acid mutations topromote heterodimer formation with increased stability, wherein thevariant CH3 domain is AZ17, or AZ18, or AZ19, or AZ20, or AZ21, or AZ22,or AZ23, or AZ24, or AZ25, or AZ26, or AZ27, or AZ28, or AZ29, or AZ30,or AZ21, or AZ32, or AZ33, or AZ34, or AZ35, or AZ36, or AZ37, or AZ38,or AZ39, or AZ40, or AZ41, or AZ42, or AZ43, or AZ44, or AZ45, or AZ46,or AZ47, or AZ48, or AZ49, or AZ50, or AZ51, or AZ52, or AZ53, or AZ54,or AZ55, or AZ56 or AZ57, or AZ58, or AZ59, or AZ60, or AZ61, or AZ62,or AZ63, or AZ64, or AZ65, or AZ66, or AZ67, or AZ68, or AZ69, or AZ70,or AZ71, or AZ72, or AZ73, or AZ74, or AZ75, or AZ76, or AZ77, or AZ78,or AZ79, or AZ80, or AZ81, or AZ82, or AZ83, or AZ84, or AZ85, or AZ86,or AZ87, or AZ88, or AZ89, or AZ90, or AZ91, or AZ92, or AZ93, or AZ94,or AZ95, or AZ96, or AZ97, or AZ98, or AZ99, or AZ100 or AZ101. In anexemplary embodiment, the variant CH3 domain is AZ17, or AZ18, or AZ19,or AZ20, or AZ21, or AZ22, or AZ23, or AZ24, or AZ25, or AZ26, or AZ27,or AZ28, or AZ29, or AZ30, or AZ21, or AZ32, or AZ33, or AZ34, or AZ38,or AZ42, or AZ43, or AZ 44, or AZ45, or AZ46, or AZ47, or AZ48, or AZ49,or AZ50, or AZ52, or AZ53, or AZ54, or AZ58, or AZ59, or AZ60, or AZ61,or AZ62, or AZ63, or AZ64, or AZ65, or AZ66, or AZ67, or AZ68, or AZ69,or AZ70, or AZ71, or AZ72, or AZ73, or AZ74, or AZ75, or AZ76, or AZ77,or AZ78, or AZ79, or AZ81, or AZ82, or AZ83, or AZ84, or AZ85, or AZ86,or AZ87, or AZ88, or AZ89, or AZ91, or AZ92, or AZ93, or AZ94, or AZ95,or AZ98, or AZ99, or AZ100 or AZ101. In a specific embodiment, thevariant CH3 domain is AZ33 or AZ34. In another embodiment, the variantCH3 domain is AZ70 or AZ90.

In an exemplary embodiment, the CH3 domain comprises a first and secondpolypeptide (also referred to herein as Chain A and Chain B) wherein thefirst polypeptide comprises amino acid modifications L351Y, F405A, andY407V and wherein the second polypeptide comprises amino acidmodifications T366I, K392M and T394W. In another embodiment, a firstpolypeptide comprises amino acid modifications L351Y, S400E, F405A andY407V and the second polypeptide comprises amino acid modificationsT366I, N390R, K392M and T394W.

This iterative process of computational structure-function analysis,targeted engineering and experimental validation was used to design theremaining Fc variants listed in Table 1 in subsequent design phases andresulting in Fc variant heterodimers with a purity greater than 90% andan increased stability with a CH3 domain melting temperature greaterthan 70° C. In certain embodiments, the Fc variants comprise amino acidmutations selected from AZ1 to AZ 136. In further embodiments, the Fcvariants comprise amino acid mutations selected from the Fc variantslisted in Table 4.

From the first and second design phases two core scaffolds wereidentified, Scaffold 1 and Scaffold 2, wherein additional amino acidmodifications were introduced into these scaffolds to fine tune thepurity and stability of the Fc variant heterodimers. See Example 5 for adetailed description of the development of Scaffold 1 including AZ8,AZ17-62 and the variants listed in Table 6. See Example 6 for a detaileddescription of the development of Scaffold 2 including AZ15 and AZ63-101and the variants listed in Table 7.

The core mutations of Scaffold 1 comprise L351Y_F405A_Y407V/T394W.Scaffold 1a comprises the amino acid mutationsT366I_K392M_T394W/F405A_Y407V and Scaffold 1b comprises the amino acidmutations T366L_K392M_T394W/F405A_Y407V. See, Example 5.

In certain embodiments, the variant CH3 domain comprises a first andsecond polypeptide (also referred to herein as Chain A and Chain B)wherein the first polypeptide comprises amino acid modifications L351Y,F405A and Y407V and the second polypeptide comprises amino acidmodification T394W. In one aspect the variant CH3 domain furthercomprises point mutations at positions F405 and/or K392. These mutationsat position K392 include, but are not limited to, K392V, K392M, K392R,K392L, K392F or K392E. These mutations at position F405 include, but arenot limited to, F405I, F405M, F405S, F405S, F405V or F405W. In anotheraspect, the variant CH3 domain further comprises point mutations atpositions T411 and/or S400. These mutations at position T411 include,but are not limited to, T411N, T411R, T411Q, T411K, T411D, T411E orT411W. These mutations at position S400 include, but are not limited to,S400E, S400D, S400R or S400K. In yet another embodiment, the variant CH3domain comprises a first and second polypeptide wherein the firstpolypeptide comprises amino acid modifications L351Y, F405A and Y407Vand the second polypeptide comprises amino acid modification T394W,wherein the first and/or second polypeptide comprises further amino acidmodifications at positions T366 and/or L368. These mutations at positionT366 include, but are not limited to, T366A, T366I, T366L, T366M, T366Y,T366S, T366C, T366V or T366W. In an exemplary embodiment, the amino acidmutation at position T366 is T366I. In another exemplary embodiment, theamino acid mutation at position T366 is T366L. The mutations at positionL368 include, but are not limited to, L368D, L368R, L368T, L368M, L368V,L368F, L368S and L368A.

In certain embodiments, the variant CH3 domain comprises a first andsecond polypeptide (also referred to herein as Chain A and Chain B)wherein the first polypeptide comprises amino acid modifications L351Y,F405A and Y407V and the second polypeptide comprises amino acidmodifications T366L and T394W. In another embodiment, the variant CH3domain comprises a first and second polypeptide wherein the firstpolypeptide comprises amino acid modifications L351Y, F405A and Y407Vand the second polypeptide comprises amino acid modifications T366I andT394W.

In certain other embodiments, the variant CH3 domain comprises a firstand second polypeptide (also referred to herein as Chain A and Chain B)wherein the first polypeptide comprises amino acid modifications L351Y,F405A and Y407V and the second polypeptide comprises amino acidmodifications T366L, K392M and T394W. In another embodiment, the variantCH3 domain comprises a first and second polypeptide wherein the firstpolypeptide comprises amino acid modifications L351Y, F405A and Y407Vand the second polypeptide comprises amino acid modifications T366I,K392M and T394W.

In yet another embodiment, the variant CH3 domain comprises a first andsecond polypeptide (also referred to herein as Chain A and Chain B)wherein the first polypeptide comprises amino acid modifications F405Aand Y407V and the second polypeptide comprises amino acid modificationsT366L, K392M and T394W. In another embodiment, the variant CH3 domaincomprises a first and second polypeptide wherein the first polypeptidecomprises amino acid modifications F405A and Y407V and the secondpolypeptide comprises amino acid modifications T366I, K392M and T394W.

In certain embodiments, the variant CH3 domain comprises a first andsecond polypeptide (also referred to herein as Chain A and Chain B)wherein the first polypeptide comprises amino acid modifications F405Aand Y407V and the second polypeptide comprises amino acid modificationsT366L and T394W. In another embodiment, the variant CH3 domain comprisesa first and second polypeptide wherein the first polypeptide comprisesamino acid modifications F405A and Y407V and the second polypeptidecomprises amino acid modifications T366I and T394W.

In an exemplary embodiment, provided herein are isolated heteromultimerscomprising a heterodimer Fc region, wherein the heterodimer Fc regioncomprises a variant CH3 domain comprising amino acid mutations topromote heterodimer formation with increased stability, wherein thevariant CH3 domain has a melting temperature (Tm) of about 74° C. orgreater. In another embodiment, provided herein are isolatedheteromultimers comprising a heterodimer Fc region, wherein theheterodimer Fc region comprises a variant CH3 domain comprising aminoacid mutations to promote heterodimer formation with increasedstability, wherein the variant CH3 domain has a melting temperature (Tm)of about 74° C. or greater and the heterodimer has a purity of about 98%or greater.

In certain embodiments, the isolated heteromultimer comprising aheterodimer Fc region, wherein the heterodimer Fc region comprises avariant CH3 domain comprising amino acid mutations to promoteheterodimer formation with increased stability, wherein the variant CH3domain has a melting temperature (Tm) greater than 70° C. and thevariant CH3 domains are selected from Table 6.

The core mutations of Scaffold 2 comprise L351Y_Y407A/T366A_K409F.Scaffold 2a comprises the amino acid mutations L351Y_Y407A/T366V_K409Fand Scaffold 2b comprises the amino acid mutations Y407A/T366A_K409F.See, Example 6.

In certain embodiments, the variant CH3 domain comprises a first andsecond polypeptide (also referred to herein as Chain A and Chain B)wherein the first polypeptide comprises amino acid modifications L351Yand Y407A and the second polypeptide comprises amino add modificationsT366A and K409F. In one aspect the variant CH3 domain further comprisespoint mutations at positions T366, L351, and Y407. These mutations atposition T366 include, but are not limited to, T366I, T366L, T366M,T366Y, T366S, T366C, T366V or T366W. In a specific embodiment, themutation at position T366 is T366V. The mutations at position L351include, but are not limited to, L351I, L351D, L351R or L351F. Themutations at position Y407 include, but are not limited to, Y407V orY407S. See, CH3 variants AZ63-AZ70 in Table 1 and Table 4 and Example 6.

In an exemplary embodiment, the variant CH3 domain comprises a first andsecond polypeptide (also referred to herein as Chain A and Chain B)wherein the first polypeptide comprises amino acid modifications L351Yand Y407A and the second polypeptide comprises amino acid modificationT366V and K409F.

In an exemplary embodiment, provided herein are isolated heteromultimerscomprising a heterodimer Fc region, wherein the heterodimer Fc regioncomprises a variant CH3 domain comprising amino acid mutations topromote heterodimer formation with increased stability, wherein thevariant CH3 domain has a melting temperature (Tm) of about 75.5° C. orgreater. In another embodiment, provided herein are isolatedheteromultimers comprising a heterodimer Fc region, wherein theheterodimer Fc region comprises a variant CH3 domain comprising aminoacid mutations to promote heterodimer formation with increasedstability, wherein the variant CH3 domain has a melting temperature (Tm)of about 75° C. or greater and the heterodimer has a purity of about 90%or greater.

In other certain embodiments, the variant CH3 domain comprises a firstand second polypeptide (also referred to herein as Chain A and Chain B)wherein the first polypeptide comprises amino acid modifications L351Yand Y407A and the second polypeptide comprises amino acid modificationT366A and K409F, wherein the variant CH3 domain comprises one or moreamino acid modifications at positions T411, D399, 5400, F405, N390,and/or K392. These mutations at position D399 include, but are notlimited to, D399R, D399W, D399Y or D399K. The mutations at position T411includes, but are not limited to, T411N, T411R, T411Q, T411K, T411D,T411E or T411W. The mutations at position S400 includes, but are notlimited to, S400E, S400D, S400R, or 3400K. The mutations at positionF405 includes, but are not limited to, F405I, F405M, F405S, F405S, F405Vor F405W. The mutations at position N390 include, but are not limitedto, N390R, N390K or N390D. The mutations at position K392 include, butare not limited to, K392V, K392M, K392R, K392L, K392F or K392E. See, CH3variants AZ71-101 in Table 1 and Table 4 and Example 6.

In an exemplary embodiment, the variant CH3 domain comprises a first andsecond polypeptide (also referred to herein as Chain A and Chain B)wherein the first polypeptide comprises amino acid modification Y407Aand the second polypeptide comprises amino acid modification T366A andK409F. In one aspect, this variant CH3 domain further comprises theamino acid modifications K392E, T411E, D399R and S400R. In a furtherembodiment, the variant CH3 domain comprises a first and secondpolypeptide wherein the first polypeptide comprises amino acidmodification D399R, S400R and Y407A and the second polypeptide comprisesamino acid modification T366A, K409F, K392E and T411E. In an exemplaryembodiment, provided herein are isolated heteromultimers comprising aheterodimer Fc region, wherein the heterodimer Fc region comprises avariant CH3 domain comprising amino acid mutations to promoteheterodimer formation with increased stability, wherein the variant CH3domain has a melting temperature (Tm) of about 74° C. or greater. Inanother embodiment, provided herein are isolated heteromultimerscomprising a heterodimer Fc region, wherein the heterodimer Fc regioncomprises a variant CH3 domain comprising amino acid mutations topromote heterodimer formation with increased stability, wherein thevariant CH3 domain has a melting temperature (Tm) of about 74° C. orgreater and the heterodimer has a purity of about 95% or greater.

In certain embodiments, provided herein are isolated heteromultimerscomprising a heterodimer Fc region, wherein the heterodimer Fc regioncomprises a variant CH3 domain comprising amino acid mutations topromote heterodimer formation with increased stability, wherein thevariant CH3 domain has a melting temperature (Tm) greater than 70° C.and the variant CH3 domains are selected from Table 7.

Furthermore, this new method of designing Fc variant heterodimers withimproved stability and purity can be applied to other classes andisotypes of Fc regions. In certain embodiments, the Fc region is a humanIgG Fc region. In further embodiments, the human IgG Fc region is ahuman IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments the Fcregions is from an immunoglobulin selected from the group consisting ofIgG, IgA, IgD, IgE and IgM. In some embodiments, the IgG is of subtypeselected from the group consisting of IgG1, IgG2a, IgG2b, IgG3 and IgG4.

TABLE 1 CH3 domain amino acid modifications for the generation of Fcvariant heterodimers. Variant Chains Fc Mutations Wild- A — — — — — — —Type B — — — — — — — IgG1 CH3 AZ1 A L368D K370Q — — — — — Variants BE357R L368R — — — — — AZ2 A L3511I L368D K370Q — — — — B E357R L368R — —— — — AZ3 A L351D L368D K370Q — — — — B E357R L368R — — — — AZ4 A L368DK370E — — — — — B E357R L368R — — — — — AZ5 A L368D K370E — — — — — BE357K L368R — — — — — AZ6 A V397S F405A Y407V — — — — B K392V T394W — —— — — AZ7 A L351R V397S F405A Y407V — — — B K392V T394W — — — — — AZ8 AL351Y V397S F405A Y407V — — — B K392V T394W — — — — — AZ9 A V397S F405AY407V — — — — B L368R K392V T394W — — — — AZ10 A V397T F405I — — — — — BK392V T394H — — — — — AZ11 A E357W S364F — — — — — B Y349A L351Y K370I —— — — AZ12 A E357H S364F — — — — — B L351Y K370I — — — — — AZ13 A E357WS364F — — — — — B Y349A L351Y K370F — — — — AZ14 A E357H S364F — — — — —B L351Y K370F — — — — — AZ15 A E357L T366A K409F T411N — — — B L351YY407A — — — — — AZ16 A E357L T366A K409Y T411N — — — B L351Y L368T Y407A— — — — AZ17 A L351Y F405A Y407V — — — — B T366I T394W — — — — — AZ18 AL351Y V397T F405M Y407V — — — B T366I T394W — — — — — AZ19 A L351Y V397TF405M Y407V — — — B T366L T394W — — — — — AZ20 A L351Y V397T F405M Y407V— — — B T366M T394W — — — — — AZ21 A L351Y L368M V397T F405I Y407V — — BT366L T394W — — — — — AZ22 A L351Y L368M V397T F405I Y407V — — B T366MT394W — — — — — AZ23 A L351Y V397T F405M Y407V — — — B L351I T366I T394W— — — AZ24 A L351Y V397T L398D F405M Y407V — — B S354E T366I T394W — — —— AZ25 A L351Y V397T L398D S400E F405M Y407V — B T366I N390R T394W — — —— AZ26 A R344H L351Y V397T S400E F405M Y407V — B Q362R T366I T394W — — —— AZ27 A R344H L351Y V397T D401E F405M Y407V — B Q362R T366I T394W — — —— AZ28 A Q347R L351Y V397T F405M Y407V — — B S354E K360E T366I T394W — —— AZ29 A Q347R L351Y V397T F405M Y407V — — B S354N K360E T366I T394W — —— AZ30 A T350V L351Y V397T S400E F405M Y407V — B T350V T366I T394W T411R— — — AZ31 A R344H L351Y V397T L398D F405M Y407V — B T366I T394W T411R —— — — AZ32 A Q347R T350V L351Y V397T F405M Y407V — B T350V K360E T366IT394W T411R — — AZ33 A L351Y F405A Y407V — — — — B T366I K392M T394W — —— — AZ34 A L351Y S400E F405A Y407V — — — B T366I N390R K392M T394W — — —AZ35 A L351Y K370Q G371D F405M Y407V — — B Q362R T366I T394W K409R T411Q— — AZ36 A L351Y K370Q G371D F405S Y407V — — B Q362R T366I T394W K409RT411Q — — AZ37 A R344H L351Y K370Q G371D L398D F405M Y407V B Q362R T366IT394W K409R T411Q — — AZ38 A R344H L351Y K370Q G371D S400E F405M Y407V BQ362R T366I N390R T394W K409R T411Q — AZ39 A L351Y K370Q G371D F405MY407V — — B T366I T394W T411R — — — — AZ40 A L351Y K370Q G371D F405MY407V — — B T366I T394W K409M T411R — — — AZ41 A R344H L351Y K370Q G371DL398D F405M Y407V B T366I T394W K409M T411R — — — AZ42 A R344H L351YK370Q G371D S400E F405M Y407V B T366I N390R T394W K409M T411R — — AZ43 AL351Y K370T G371D F405I Y407V — — B E357Q S364R T394W — — — — AZ44 AL351Y K370T G371D F405M Y407V — — B E357Q S364R T394W K409I — — — AZ45 AR344H L351Y K370T G371D S400E F405M Y407V B E357Q S364R T366I N390RT394W K409I — AZ46 A R344H L351Y K370T G371D F405M Y407V — B E357Q S364RT366I T394W K409I T411R — AZ47 A L351Y K370A G371S D399R F405S Y407V — BE357Q Q362R T364Y T366I T394W K409S — AZ48 A L351Y V397S D399W F405MY407V — — B Q362R T366I T394W K409M — — — AZ49 A L351Y V397S D399Y F405MY407V — — B Q362R T366I T394W K409I — — — AZ50 A R344H L351Y V397T L398DD399W F405M Y407V B Q362R T366I T394W K409M — — — AZ51 A R344H L351YV397T D399W S400E F405M Y407V B Q362R T366I T394W K409M — — — AZ52 AL368V K370F F405I Y407V — — — B E357Q S364Y T366I T394W — — — AZ53 AL368V K370Y F405I Y407V — — — B E357Q S364Y T394W — — — — AZ54 A R344HL368V K370Y F405M Y407V — — B E357Q Q362R S364Y T394W — — — AZ55 A L368VK370Y S400E F405M Y407V — — B E357Q S364Y N390R T394W — — — AZ56 A L368VK370Y L398D F405M Y407V — — B E357Q S364Y T394W T411R — — — AZ57 A R344HL351Y K370Y F405M Y407V — — B E357Q Q362R T364T T366I T394W — — AZ58 AL368V V397T F405M Y407V — — — B T366Y T394W — — — — — AZ59 A L368V K370QV397T F405M Y407V — — B T366Y T394W — — — — — AZ60 A R344H L368V V397TS400E F405M Y407V — B Q362R T366Y T394W — — — — AZ61 A L368V V397T S400EF405M Y407V — — B T366Y N390R T394W — — — — AZ62 A L368V V397T L398DF405M Y407V — — B T366Y T394W T411R — — — — AZ63 A T366A K409F — — — — —B Y407A — — — — — — AZ64 A T366A K409F — — — — — B L351Y Y407A — — — — —AZ65 A T366A K409F — — — — — B L351F Y407A — — — — — AZ66 A T366S K409F— — — — — B Y407A — — — — — — AZ67 A T366C K409F — — — — — B Y407A — — —— — — AZ68 A T366L K409F — — — — — B Y407A — — — — — — AZ69 A T366MK409F — — — — — B Y407A — — — — — — AZ70 A T366V K409F — — — — — B L351YY407A — — — — — AZ71 A T366A K409F — — — — — B L351I T366S L368F Y407A —— — AZ72 A T366A K409F — — — — — B D399W Y407A — — — — — AZ73 A T366AK409F — — — — — B D399W S400D Y407A — — — — AZ74 A T366A K409F — — — — —B D399W S400E Y407A — — — — AZ75 A T366A K409F T411R — — — — B D399WS400D Y407A — — — — AZ76 A T366A K409F T411R — — — — B G371D D399W Y407A— — — — AZ77 A T366A K409F T411R — — — — B K370Q G371D D399W Y407A — — —AZ78 A T366A N390R K409F — — — — B D399Y S400D Y407A — — — — AZ79 AQ362R T366A K409F T411K — — — B Y407A — — — — — — AZ80 A Q362R T366AK409F T411R — — — B Y407A — — — — — — AZ81 A Q362K T366A K409F T411R — —— B Y407A — — — — — — AZ82 A T366A N390K K392R K409F T411R — — B S400EY407A — — — — — AZ83 A T366A N390K K392R K409F T411K — — B S400E Y407A —— — — — AZ84 A T366A N390K K409F T411R — — — B S400D Y407A — — — — —AZ85 A T366A K392L K409F T411D — — — B D399R Y407A — — — — — AZ86 AT366A K392L K409F T411E — — — B D399R Y407A — — — — — AZ87 A T366A K392LK409F T411D — — — B D399K Y407A — — — — — AZ88 A T366A K392L K409F T411E— — — B D399K Y407A — — — — — AZ89 A T366A K392M K409F T411E — — — BD399R Y407A — — — — — AZ90 A T366A K392M K409F T411D — — — B D399R Y407A— — — — — AZ91 A T366A K392F K409F T411D — — — B D399R F405V Y407A — — —— AZ92 A T366A K409F T411E — — — — B D399R S400E Y407A — — — — AZ93 AT366A K409F T411E — — — — B D399R S400D Y407A — — — — AZ94 A T366A K392EK409F T411E — — — B D399R S400R Y407A — — — — AZ95 A T366A K392E K409FT411D — — — B D399R S400R Y407A — — — — AZ96 A Q362E T366A K409F T411W —— — B D399R Y407A — — — — — AZ97 A Q362D T366A K409F T411W — — — B D399RY407A — — — — — AZ98 A S364Y T366A K409F T411R — — — B Y407A — — — — — —AZ99 A T366V K409W — — — — — B L368V Y407S — — — — — AZ100 A T366V K409W— — — — — B L351Y L368S Y407A — — — — AZ101 A T366V K409W — — — — — BL351Y Y407A — — — — — AZ102 A E357Q S364F K392E — — — — B K370F V397RS400R — — — — AZ103 A E357Q S364F K392E V397E — — — B K370F V397R S400R— — — — AZ104 A E357Q S364F N390D K392E — — — B K370F V397R S400K — — —— AZ105 A E357Q S364F K370E G371W — — — B E357Q K360R S364N K370F — — —AZ106 A S354R D356K E357Q S364F — — — B S354E K370F K439E — — — — AZ107A Q347R E357Q S364F — — — — B Q347E K360E K370F — — — — AZ108 A E357QS364F K370E — — — — B E357R K370F — — — — — AZ109 A E357Q S364F L368DK370E — — — B E357R K370F — — — — — AZ110 A E357Q S364F K370T G371D — —— B E357Q S364R K370F — — — — AZ111 A E357Q S364Y K392E — — — — B K370FV397R S400K — — — — AZ112 A E357Q S364Y K392E — — — — B L368A K370FV397R S400K — — — AZ113 A K409F T411E — — — — — B L368V D399R S400D — —— — AZ114 A K409F T411E — — — — — B L368V D399K S400D — — — — AZ115 AK409F — — — — — — B L368V D399Y — — — — — AZ116 A E357Q K409F T411R — —— — B L368A K370F — — — — — AZ117 A S354R D356K K409F T411R — — — BS354E L368V S400E K439E — — — AZ118 A K360E K370E — — — — — B Y349RE357R — — — — — AZ119 A K360E K370E — — — — — B Y349K E357R — — — — —AZ120 A S354E K360E K370E — — — — B Y349R E357R — — — — — AZ121 A K360EL368D K370E — — — — B Y349R E357R — — — — — AZ122 A K360E L368D K370E —— — — B Y349R E357R T411R — — — — AZ123 A K360E K370T G371D — — — — BY349R E357Q S364R — — — — AZ124 A K360E K370T G371D — — — — B Y349RE357Q S364K — — — — AZ125 A S364E K370T G371D — — — — B E357Q S364RG371R — — — — AZ126 A S364E K370T G371D — — — — B E357Q S364R G371K — —— — AZ127 A G371D T411E — — — — — B G371R T411R — — — — — AZ128 A G371DT411E — — — — — B G371K T411R — — — — — AZ129 A Y349C L351Y V397T F405MY407V — — B S354C T366I T394W — — — — AZ130 A L351Y S354C V397T F405MY407V — — B Y349C T366I T394W — — — — AZ132 A L368A F405W Y407V — — — —B T366W — — — — — —

The Fc region as defined herein comprises a CH3 domain or fragmentthereof, and may additionally comprise one or more addition constantregion domains, or fragments thereof, including hinge, CH1, or CH2. Itwill be understood that the numbering of the Fc amino acid residues isthat of the EU index as in Kabat et al., 1991, NIH Publication 91-3242,National Technical Information Service, Springfield, Va. The “EU indexas set forth in Kabat” refers to the EU index numbering of the humanIgG1 Kabat antibody. For convenience, Table B provides the amino acidsnumbered according to the EU index as set forth in Kabat of the CH2 andCH3 domain from human IgG1.

TABLE B CH2 Domain CH3 Domain EU Amino EU Amino EU Amino EU Amino EUAmino EU Amino No. Acid No. Acid No. Acid No. Acid No. Acid No. Acid 231A 271 P 311 Q 341 G 381 W 421 N 232 P 272 E 312 D 342 Q 382 E 422 V 233E 273 V 313 W 343 P 383 S 423 F 234 L 274 K 314 L 344 R 384 N 424 S 235L 275 F 315 N 345 E 385 G 425 C 236 G 276 N 316 G 346 P 386 Q 426 S 237G 277 W 317 K 347 Q 387 P 427 V 238 P 278 Y 318 E 348 V 388 E 428 M 239S 279 V 319 Y 349 Y 389 N 429 H 240 V 280 D 320 K 350 T 390 N 430 E 241F 281 G 321 C 351 L 391 Y 431 A 242 L 282 V 322 K 352 P 392 K 432 L 243F 283 E 323 V 353 P 393 T 433 H 244 P 284 V 324 S 354 S 394 T 434 N 245P 285 H 325 N 355 R 395 P 435 H 246 K 286 N 326 K 356 D 396 P 436 Y 247P 287 A 327 A 357 E 397 V 437 T 248 K 288 K 328 L 358 L 398 L 438 Q 249D 289 T 329 P 359 T 399 D 439 K 250 T 290 K 330 A 360 K 400 S 440 S 251L 291 P 331 P 361 N 401 D 441 L 252 M 292 R 332 I 362 Q 402 G 442 S 253I 293 E 333 E 363 V 403 S 443 L 254 S 294 E 334 K 364 S 404 F 444 S 255R 295 Q 335 T 365 L 405 F 445 P 256 T 296 Y 336 I 366 T 406 L 446 G 257P 297 N 337 S 367 C 407 Y 447 K 258 E 298 S 338 K 368 L 408 S 259 V 299T 339 A 369 V 409 K 260 T 300 Y 340 K 370 K 410 L 261 C 301 R 371 G 411T 262 V 302 V 372 F 412 V 263 V 303 V 373 Y 413 D 264 V 304 S 374 P 414K 265 D 305 V 375 S 415 S 266 V 306 L 376 D 416 R 267 S 307 T 377 I 417W 268 H 308 V 378 A 418 Q 269 E 309 L 379 V 419 Q 270 D 310 H 380 E 420G

In certain embodiments, the Fc variant comprises a CH2 domain. In someembodiments, the CH2 domain is a variant CH2 domain. In someembodiments, the variant CH2 domains comprise asymmetric amino acidsubstitutions in the first and/or second polypeptide chain. In someembodiments, the heteromultimer comprises asymmetric amino acidsubstitutions in the CH2 domain such that one chain of saidheteromultimer selectively binds an Fc receptor.

In certain embodiments, the heteromultimer selectively binds an Fcreceptor. In some embodiments, Fc receptor is a member of Fcγ receptorfamily. In some embodiments, the receptor is selected from FcγRI,FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and FcγRIIIb. In one embodiment, theCH2 domain comprises asymmetric amino acid modifications that promoteselective binding to Fcgamma receptors.

In some embodiments, the heteromultimer binds selectively to FcγRIIIa.In some embodiments, the heteromultimer comprises asymmetric amino acidsubstitutions selected from S267D, K392D and K409D. In some embodiments,the heteromultimer binds selectively to FcγRIIa. In some embodiments,the heteromultimer comprises asymmetric amino acid substitutionsselected from S239D, K326E, A330L and I332E. In some embodiments, theheteromultimer binds selectively to FcγRIIb. In some embodiments, theheteromultimer comprises asymmetric amino acid substitutions selectedfrom S239D, D265S, E269K and I332E. In some embodiments, theheteromultimer binds selectively to FcγRIIIa and FcγRIIa. In someembodiments, the heteromultimer comprises asymmetric amino acidsubstitutions selected from S239D, D265S, and S298A. In someembodiments, the heteromultimer binds selectively to FcγRIIIa andFcγRIIb. In some embodiments, the heteromultimer comprises asymmetricamino acid substitutions selected from S239D, S298A, K326E, A330L andI332E. In some embodiments, the heteromultimer binds selectively toFcγRIIa and FcγRIIb. In some embodiments, the heteromultimer comprisesasymmetric amino acid substitutions selected from S239D, D265S, S298Aand I332E.

In certain embodiments is a method of designing multi-functionaltherapeutics comprising heteromultimer described herein. In someembodiments is method of designing bi-functional therapeutics comprisinga variant Fc heterodimer. In some embodiments is a method for the designof asymmetric mutations in the CH2 domain of a variant Fc heterodimerderived with mutations in the CH3 domain. In some embodiments is amethod to design selectivity for the different Fc gamma receptors basedon the mutations in the asymmetric Fc. In certain embodiments is amethod to design mutations that bias binding of the Fc gamma receptorsto one face of the Fc molecule. In certain embodiments is a method todesign polarity drivers that bias the Fcγ receptors to interact withonly one face of the asymmetric Fc scaffold of the heteromultimerdescribed herein.

In some embodiments, is provided a polypeptide comprising mutations inthe CH2 domain of the asymmetric Fc that lead to preferential Fc gammareceptor selectivity profiles. In some embodiments mutations in the CH3domain lead to preferential formation of heterodimeric Fc. In certainembodiments is a method for designing bispecific therapeutic entitiesbased on the asymmetric Fc described herein. In certain embodiments is amethod to design multi-specific therapeutic entities based on theasymmetirc Fc described herein.

Monoclonal antibodies such as IgG are symmetric molecules composed oftwo equivalent heavy and two light polypeptide chains (FIG. 14), eachcomprising multiple immunoglobulin (Ig) structural domains. The IgGclass of mAb's exists in one of four isoforms, IgG1, IgG2, IgG3, orIgG4. The heavy chain is composed of four (VH, CH1, CH2 and CH3) and thelight chain of two (VL and CL) Ig domains, respectively. The VH and CH1domains from each of the heavy chains combine with the VL and CL domainsof light chain to form the two Fab (“fragment antigen binding”) arms ofthe mAb. The CH3 and CH2 domains of the two heavy chains interact viaprotein-protein contacts across the CH3 domains and glycosylation in theCH2 domains to form the homodimeric Fc (“fragment crystallizable”)region. The linker region between CH1 and CH2 domains of the antibodyconstitutes the hinge region of the antibody molecule. Apart fromconnecting the Fab and Fc regions of the mAb, the hinge also maintainsdisulphide links across the two heavy chains and holds them together.The number of amino acids and disulphide links in the hinge region isnotably different among the four isotypes of IgG. The glycosylationpattern in IgG molecules can be significantly diverse, about 30different carbohydrate moieties have been observed in IgG molecules[Arnold J. N.; Wormald M. R.; Sim R. B.; Rudd P. M. and Dwek R. A.(2007) Annual Reviews of Immunology 25, 21-50].

The symmetric nature of the monoclonal antibodies structure results inboth Fab arms having their antigen binding capability affinity maturedto recognize the same epitope. At the other end, the Fc portion of theantibody molecule is involved in interactions with various receptormolecules on the immune or “effector” cells, and some of theseinteractions are responsible for mediating effector functions such asantibody dependent cellular cytotoxicity (ADCC), antibody dependentcellular phagocytosis (ADCP) and complement activation. Generally, theeffector function involves immune responses leading to pathogen or toxinneutralization and elimination, complement activation, and phagocyticresponse from the humoral immune system. The Fcγ receptor (FcγR)molecules on the effector cells contact the Fc of the activated IgGantibody involved in integral antibody-antigen immune complex to mediateand regulate the effector response. Optimizing the interaction ofmonoclonal antibody based protein therapeutic agents to these Fcγreceptors can lead to improvements in the efficacy of these drugcandidates.

In humans there are three known classes of FcγR's with furtherpolymorphic types within each class. The Fc in the IgG1 molecule isknown to bind FcγRI (CD64) with dissociation constants in the nanomolarrange while FcγRII (CD32) and fcγRIII (CD16) binding occurs at themicromolar range [Bruhns P.; Iannascoli B.; England P.; Mancardi D. A.;Fernandez N.; Jorieux S, and Daeron M. (2009) Blood 113: 3716-25]. Thehigh affinity FcγRI receptors can bind IgG in monomeric forms while thelow affinity FcγRII and FcγRIII receptors can only bind antigen-antibodyimmune complexes or IgG aggregates as a result of avidity effects. Thedifferent IgG forms have varying affinities for the different FcγR's; inparticular, the IgG1 and IgG3 exhibit stronger activity. The Feyreceptors are the extracellular domains of trans-membrane proteins andpossess cytoplasmic domains that are involved in regulating signalingpathways within the cell. When clustered on the immune cell surface onassociation with the antibody mediated immune complexes, depending onthe nature of signaling units linked to the FcγR's on the cytoplasmicend of these cell surface receptors, these molecules regulate theeffector response [Nimmerjahn F. and Ravetch J. V. (2008) Nature ImmuRev 8(1):34-47].

At the human chromosomal level, three genes encode the FcγRI (FcγRIA,FcγRIB, FcγRIC) and FcγRII (FcγRIIA, FcγRIIB, FcγRIIC) and two genesencode the FcγRIII (FcγRIIIA, FcγRIIIB). Among the IgG binding human Feyreceptors, the FcγRIA, FcγRIC and FcγRIIIA types have been shown to bemembrane associated with a common γ-chain signal adaptor protein whichcontains a cytoplasmic immunoreceptor tyrosine based activation motif(ITAM) that leads to the activation of effector function. The FcγRIIAand FcγRIIC also comprise a cytoplasmic ITAM, but without the commonγ-chain signal adaptor protein. At the same time, the FcγRIIB is linkedto an immunoreceptor tyrosine-based inhibitory motif (ITIM). Activationof FcγRIIB resulting in ITIM phosphorylation results in inhibition ofthe activating signaling cascade. The FcγRIIIB, while lacking either ofthe tyrosine based immuno-modulatory cytoplasmic tails, has a GPI(glycosyl-phosphatidyl-inositol) anchor and has been shown to contributeto activation of some granulocytes in the presence of FcγRIIA.

TABLE C Fcγ Receptor Characteristics Signaling Receptor Alleles MotifFunction IgG Binding Affinity FcγRI ITAM Activating IgG1 ≈ IgG3 > IgG4(CD64) FcγRIIa 131(H/R) ITAM Activating IgG1 > IgG3 > IgG2 > (CD32a)IgG4 FcγRIIb 232(I/T) ITIM Inhibitory IgG3 ≈ IgG1 ≈ (CD32b) IgG4 > IgG2FcγRIIc 57(Q/ ITAM Activating IgG3 ≈ IgG1 ≈ (CD32c) Trunca- IgG4 > IgG2tion) FcγRIIIa 158(V/F) ITAM Activating IgG3 > IgG1 > IgG4 > (CD16a)IgG2 FcγRIIIb NA1/2 GPI Activating IgG3 > IgG1 (CD16b) SH/78(A/D) ITAM:Immune-receptor Tyrosine based Activation Motif; ITIM: Immuno-receptorTyrosine based Inhibition Motif; GPI Glycophosphoinositol

While the functional role of ITAM and ITIM motifs and the associatedreceptor molecules are known, the nature and mechanisms of themodulation of signaling in combination is not completely understood,especially when combined with the activity of a host of other immunecell surface receptors and adaptor molecules (e.g. BCR's, CD22, CD45etc) involved in signal transduction. In this context, the design ofFc-like molecules that can interact with these Fcγ receptors withexquisite selectivity profiles is a valuable scaffold in any attempt tode-convolute and modulate the effect of such receptor molecules withsubtle regulatory activities.

In the context of designing antibody molecules that can differentiatethe FcγR's, the effort is complicated by the fact that the extracellularFc binding sections of the FcγRII and FcγRIII receptor types exhibithigh sequence similarity (FIG. 15), which can be attributed at least inpart to ancestral segmental duplication. The two major types of FcγRIIreceptors, A and B, have 69% sequence identity while the FcγRIIA andFcγRIIIA exhibit about 44% sequence identity. The FcγRIIB and FcγRIICdiffer by only 2 residues in the extracellular region, although they aresignificantly different in the intracellular region, notable being thepresence of ITIM and ITAM motifs respectively. As a result it can beanticipated that therapeutic antibody molecules required to bind onereceptor would also potentially bind to other receptor classes, possiblyresulting in unintended therapeutic effects.

Complicating matters further, each of the receptor class presentsmultiple single nucleotide polymorphisms (SNPs) and copy numbervariations (CNVs). The resulting receptor diversity differentiallyimpact their affinity to IgG's and its mechanism of action. Thesegenetic variations could affect the affinity of particular IgGsubclasses for the Fcγ receptors, alter the downstream effector eventsor impact mechanisms that alter the levels of receptor expressionresulting in functionally relevant phenotypes, non-functional orfunctionally unknown receptor variants (Bournazos S.; Woof J. M.; HartS. P. and Dransfield I. (2009) Clinical and Experimental Immunology157(2):244-54). They potentially lead to complex effects, altering thebalance between activating and inhibitory receptor signaling, resultingin the creation of disease susceptible phenotypes.

Some of these allelic variations are listed in Table C. Notably, theR131 variant in FcγRIIa is a high responder with IgG1 while thealternate H131 variants show more efficient interactions with IgG2 andIgG3. In the case of FcγRIIIa, donors homozygous for V at position 158exhibit increased NK cell activity in comparison to homozygous F/F158individuals due to higher affinity of the former allotype for humanIgG1, IgG3 and IgG4. The allelic variants NA1 and NA2 of FcγRIIIb is theresult of a four amino acid substitution which in turn leads todifferences in the glycosylation of the receptor. The NA1 allelepresents enhanced binding and phagocytosis of the immune complex byneutrophils. The FcγRIIB has two known allelic variants, 232I and 232T.The 232T variant is known to be strongly impaired in its negativeregulatory activity. The frequencies of FcγR polymorphisms and itsassociations to differential responsiveness to infections orpredisposition to disease conditions such as systemic lupus erthematosus(SLE), rheumatoid arthritis (RA), vasculitis, immune-mediatedthrombocytic purpura (ITP), myasthenia gravis, multiple sclerosis (MS),and immuno neuropathies (Guillian-Barre syndrome (GBS)) have beenreported.

Copy number variation in the locus of FcγR genes, in particular forFcγRIIIB, FcγRIIc and FcγRIIIA has been demonstrated, and furthercorrelation of these differences to cell surface expression of thesereceptors have been noted. In contrast FcγRIIa and FcγRIIb do not showgene copy number variation. Low copy number of FcγRIIIb has in fact beenassociated with glomerulonephritis in the autoimmune disease systemiclupus erythematosus (SLE) [Aitman T J et al. (2006) Nature 16;439(7078):861-5]. This is particularly interesting given the fact that anon-signaling GPI module anchors the FcγRIIIb receptor. It can behypothesized that the presence of these FcγRIIIb receptors couldpotentially act as competitive inhibitors of Fc interactions with othersignaling FcγR's. The effect of copy number variation in FcγRIIc is alsoespecially interesting. A C/T SNP at position 202 in FcγRIIc converts aglutamine residue to a stop codon preventing the generation of afunctional protein. The functional open reading frame of FcγRIIc isexpressed in 9% of healthy individuals (white population) and there is asignificant overrepresentation (19%) of the allele in the ITP populationimplying a predisposition of these phenotypes for ITP [Breunis W B etal. (2008) Blood 111(3):1029-38]. It has been demonstrated that inindividuals expressing functional FcγRIIc on NK cells, the ADCC achievedis mediated by these receptors to a greater extent than the FcγRIIIa.Such complexities associated with these polymorphisms and geneticvariations highlights the need for personalized treatment strategiesrequiring high tailored therapeutics.

The various effector cells differ in the presentation of these Fcγreceptors as well as in their humoral and tissue distribution, thuscontributing to variations in their mechanism of activation and action[Table D]. Tuning the selectivity of therapeutic antibodies towards therecognition of specific FcγR types and modulating the impact of certainclasses of effector cells, leads to optimization of the effectormechanism for particular disease conditions. This is meant toselectively activate or inhibit specific effector modalities, dependingon the disease condition being treated.

TABLE D Cellular distribution of FcγR's. FcγRI FcγRIIa FcγRIIb FcγRIIcFcγRIIIa FcγRIIIb (CD64) (CD32a) (CD32b) (CD32c) (CD16a) (CD16b)Distribution Lymphoid B cell ✓ Blood Plasma cell ✓ Tissue NK cell ✓ ✓Blood Myeloid Monocyte ✓ ✓ ✓ ✓ Blood Dendritic cell ✓ ✓ ✓ ✓ TissuePlatelet ✓ Blood Macrophage ✓ ✓ ✓ ✓ Tissue Neutrophil ✓ ✓ ✓ BloodEosinophil ✓ ✓ Blood Basophil ✓ Blood Mast cell ✓ ✓ Tissue

In addition, FcγR's are also expressed by follicular dendritic cells,endothelial cells, microglial cells, osteoclasts and mesangial cells.Currently, the functional significance of FcγR expression on these othercells is not known.

The high affinity FcγRI is composed of three C-type immunoglobulinsuperfamily (IgSF) domains while the low affinity FcγRII and FcγRIII areconstituted of two C-type IgSF domains each. The structure of FcγRIIa,FcγRIIb, FcγRIIIa and FcγRIIIb receptor proteins has been solved bycrystallography. The two IgSF domains in these structures are positioned50-55 degrees relative to each other and are connected by a hinge.

The publicly available structure of an Fc-FcγR co-complex is that of theFc-FcγRIIIb system and the FcγR geometry in the complex is maintainedvery close to that observed in the apo state of the protein [SondermannP.; Huber R.; Oosthuizen V. and Jacob U. (2000) Nature 406, 267-273.;Radaev S.; Motyaka S.; Fridman W.; Sautes-Fridman C. and Sun P. D.(2001) J Biol Chem 276, 16469-16477; Sondermann P. et al. Biochem SocTrans. 2002 August; 30(4):481-6; Sondermann P, Oosthuizen V. ImmunolLett. 2002 Jun. 3; 82(1-2):51-6; Radaev S, Sun P. Mol. Immunol. 2002May; 38(14):1073-83.] [FIG. 16]. The strong sequence and structuralsimilarity between the receptors forms the basis of comparative modelsof the Fc bound to the other receptors. On the other hand, the sequenceand structural similarity between these receptor molecules also makesthe design of Fc with the exquisite selectivity between the receptorsand their diverse isotypes challenging.

Prior to the structural evaluation of Fc-FcγR complex based oncrystallography, there were questions if the 2-fold axis of symmetry inthe Fc molecule means two potential binding sites and an effective 2:1stoichiometry for the Fc-FcγR association. Nuclear magnetic resonance(NMR) based structural studies of Fc-FcγR interactions indicate thatbinding an Fc to one FcγR on one face of the molecule induces aconformational change that precludes the binding of a second FcγRmolecule to the Fc of the same antibody molecule [Kato K. et al (2000) JMol. Biol. 295(2):213-24]. The geometry of the available co-crystalcomplex of the Fc-FcγRIIIb confirms the association of the FcγR to Fc inan asymmetric orientation with a 1:1 stoichiometry. As shown in FIG. 16,the FcγR binds to a cleft on one end of the horseshoe-shaped Fcmolecule, and is in contact with the CH2 domains from both the chains.

Alanine scanning mutagenesis [Shields R L et al. (2001) JBC 276(9):6591-604] provides insight on the residues of the Fc interfacing withthe diverse receptor types and hence involved in the Fc-FcγR interactionand recognition. Traditionally, optimization of the therapeuticantibodies has been focused around mutations that exhibit increasedbinding to the activating receptors FcγRIII [U.S. Pat. No. 6,737,056] ordecreased affinity to FcγRIIb [US2009/0010920A1]. In all these alternatevariants, mutations are introduced concurrently in both the chains.

Monoclonal antibodies often exhibit their therapeutic activity byinducing spatial localization of the target and effector immune cells. Anatural antibody mediates this by interacting with the target using itsFab domains and the effector cell using Fc domain. They are able tojuxtaposition the immune complex vis-à-vis the effector cell such thatthe cell mediated response can be induced. Avidity effects required forFcγR signaling, originating in the formation of immune complexesinvolving the targeting of a single target by multiple antibodymolecules, is another example of significance of spatio-temporalorganization in immune action.

There is also a spatio-temporal aspect to the cell signaling that isinduced as part of the effector activity of mAb molecules. Cellsignaling such as those based on FcγR molecule activation involveslocalization of the relevant receptor molecules within a region ofmembrane domain referred to as lipid rafts. Lipid rafts are enrichedwith glycosphingolipid and cholesterol and several classes of upstreamsignal transducers including the Src family kinases. Upon cellstimulation various signaling molecules, adaptor proteins and thesignaling kinases as well as phosphatases are recruited. Molecularassembly at lipid rafts is important for signal transduction.

A non-natural design strategy, combining different antigen specificitiesand increased avidity to provide better binding properties is the basisof bispecific therapeutic design. Bispecific antibodies or other formsof bispecific or multifunctional protein therapeutics are designed tomediate interactions between the target and a variety of effector cells[Müller & Kontermann (2010) BioDrugs 24(2):89-98]. Multispecifictherapeutic molecules are engineered to redirect the Helper T-cells orother immune effector cells against specific target cells.

In another embodiment, the invention relates to a method for identifyingFc variant polypeptides in silico based on calculated binding affinitiesto FcγRIIa, FcγRIIb and/or FcγRIIIa. In another embodiment, the methodfurther comprises calculating in silico electrostatics, solvation,packing, packing density, hydrogen binding, and entropic effects of saidFc variant polypeptides. In yet another embodiment, the method of thecurrent invention further includes constructing the Fc variantpolypeptides and expressing said polypeptides in the context of atherapeutic antibody and further expressing said antibody in mammaliancells. In still another embodiment the method of the current inventioncomprises constructing the Fc variant polypeptides identified in silicoby site directed mutagenesis, PCR based mutagenesis, cassettemutagenesis or de novo synthesis.

Factors taken into account in the design of the synthetic Fc scaffoldinclude in silica calculations for steric repulsion, change in buriedinterface area, relative contact density, relative salvation andelectrostatic effect. All these matrices were used to arrive at anaffinity score.

In one aspect, this application describes a molecular design forachieving exquisite FcγR selectivity profiles via the design of anasymmetric scaffold built on a heterodimeric Fc. This scaffold allowsfor asymmetric mutations in the CH2 domain to achieve a variety of novelselectivity profiles. Further, the scaffold has inherent features forthe engineering of multifunctional (bi, tri, tetra or penta functional)therapeutic molecules.

The asymmetric scaffold can be optimized for pH dependent bindingproperties to the neonatal Fc receptor (FcRn) to enable better recyclingof the molecule and enhance its half life and related pharmacokineticproperties.

The asymmetric scaffold can be optimized for binding to the functionallyrelevant FcγRI receptor allotypes. FcγRI is a prominent marker onmacrophages that are involved in chronic inflammatory disorders such asRheumatoid Arthritis, Atopic Dermatitis, Psoriasis and a number ofpulmonary diseases.

The asymmetric scaffold can be optimized for protein A binding. ProteinA binding is often employed for separation and purification of antibodymolecules. Mutations can be introduced in the asymmetric scaffold toavoid aggregation of the therapeutic during storage.

Therefore, it is specifically contemplated that the Fc variants of theinvention may contain inter alia one or more additional amino acidresidue substitutions, mutations and/or modifications which result in anantibody with preferred characteristics including but not limited to:increased serum half life, increase binding affinity, reducedimmunogenicity, increased production, enhanced or reduced ADCC or CDCactivity, altered glycosylation and/or disulfide bonds and modifiedbinding specificity.

It is contemplated that the Fc variants of the invention may have otheraltered characteristics including increased in vivo half-lives (e.g.,serum half-lives) in a mammal; in particular a human, increasedstability in vivo (e.g., serum half-lives) and/or in vitro (e.g.,shelf-life) and/or increased melting temperature (Tm), relative to acomparable molecule. In one embodiment, an Fc variant of the inventionhas an in vivo half-life of greater then 15 days, greater than 20 days,greater than 25 days, greater than 30 days, greater than 35 days,greater than 40 days, greater than 45 days, greater than 2 months,greater than 3 months, greater than 4 months, or greater than 5 months.In another embodiment, an Fc variant of the invention has an in vitrohalf-live (e.g, liquid or powder formulation) of greater then 15 days,greater than 30 days, greater than 2 months, greater than 3 months,greater than 6 months, or greater than 12 months, or greater than 24months, or greater than 36 months, or greater than 60 months.

It will also be appreciated by one skilled in the art that the Fcvariants of the invention may have altered immunogenicity whenadministered to a subject. Accordingly, it is contemplated that thevariant CH3 domain, which minimize the immunogenicity of the Fc variantare generally more desirable for therapeutic applications.

The Fc variants of the present invention may be combined with other Fcmodifications, including but not limited to modifications that altereffector function. The invention encompasses combining an Fc variant ofthe invention with other Fc modifications to provide additive,synergistic, or novel properties in antibodies or Fc fusion proteins.Such modifications may be in the hinge, CH1, or CH2, (or CH3 provided itdoes not negatively alter the stability and purity properties of thepresent variant CH3 domains) domains or a combination thereof. It iscontemplated that the Fc variants of the invention enhance the propertyof the modification with which they are combined. For example, if an Fcvariant of the invention is combined with a mutant known to bindFcγRIIIA with a higher affinity than a comparable molecule comprising awild type Fc region; the combination with a mutant of the inventionresults in a greater fold enhancement in FcγRIIIA affinity.

In one embodiment, the Fc variants of the present invention may becombined with other known Fc variants such as those disclosed in Duncanet al, 1988, Nature 332:563-564; Lund et al., 1991, J Immunol147:2657-2662; Lund et al., 1992, Mol Immunol 29:53-59; Alegre et al,1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl.Acad Sci USA 92:11980-11984; Jefferis et al, 1995, Immunol Lett.44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al, 1996,Immunol Lett 54:101-104; Lund et al, 1996, Immunol 157:4963-4969; Armouret al., 1999, Eur J Immunol 29:2613-2624; ldusogie et al, 2000, JImmunol 164; 4178-4184; Reddy et al, 2000, J Immunol 164:1925-1933; Xuet al., 2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferiset al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem SacTrans 30:487-490); U.S. Pat. Nos. 5,624,821; 5,885,573; 6,194,551; U.S.Patent Application Nos. 60/601,634 and 60/608,852; PCT Publication Nos.WO 00/42072 and WO 99/58572.

One skilled in the art will understand that the Fc variants of theinvention may have altered Fc ligand (e.g., FcγR, C1q) bindingproperties (examples of binding properties include but are not limitedto, binding specificity, equilibrium dissociation constant (K_(D)),dissociation and association rates (K_(off) and K_(on) respectively),binding affinity and/or avidity) and that certain alterations are moreor less desirable. It is well known in the art that the equilibriumdissociation constant (K_(D)) is defined as k_(off)/k_(on). It isgenerally understood that a binding molecule (e.g., and antibody) with alow K_(D) is preferable to a binding molecule (e.g., and antibody) witha high K_(D). However, in some instances the value of the k_(on) ork_(off) may be more relevant than the value of the K_(D). One skilled inthe art can determine which kinetic parameter is most important for agiven antibody application. For example a modified CH3 and/or CH2 thatenhances Fc binding to one or more positive regulators (e.g., FcγRIIIA)while leaving unchanged or even reducing Fc binding to the negativeregulator FcγRIIB would be more advantageous for enhancing ADCCactivity. Alternatively, a modified CH3 and/or CH2 that reduced bindingto one or more positive regulator and/or enhanced binding to FcγRIIBwould be advantageous for reducing ADCC activity. Accordingly, the ratioof binding affinities (e.g., equilibrium dissociation constants (K_(D)))can indicate if the ADCC activity of an Fc variant is enhanced ordecreased. For example a decrease in the ratio of FcγRIIIA/FcγRIIBequilibrium dissociation constants (K_(D)), will correlate with improvedADCC activity, while an increase in the ratio will correlate with adecrease in ADCC activity.

As part of the characterization of the Fc variants they were tested fortheir binding affinity to FcγRIIIA (CD16a) and FcγRIIB(CD32b) reportedas a ratio in comparison to wild-type IgG1. (See, Example 4 and Table 5)In this instance it was possible to evaluate the impact of the CH3domain mutations on binding to these activating and inhibitory Fcreceptors. In one embodiment, provided herein are isolatedheteromultimers comprising a heterodimer Fc region, wherein theheterodimer Fc region comprises a variant CH3 domain comprising aminoacid mutations to promote heterodimer formation with increasedstability, wherein the variant CH3 domain has a melting temperature (Tm)greater than 70° C., wherein the heterodimer binding to CD16a is aboutthe same as compared to wild-type homodimer. In certain embodiments theheterodimer binding to CD16a is increased as compared to wild-typehomodimer. In an alternative embodiment, the heterodimer binding toCD16a is reduced as compared to wild-type homodimer.

In certain embodiments, provided herein are isolated heteromultimerscomprising a heterodimer Fc region, wherein the heterodimer Fc regioncomprises a variant CH3 domain comprising amino acid mutations topromote heterodimer formation with increased stability, wherein thevariant CH3 domain has a melting temperature (Tm) greater than 70° C.,wherein the heterodimer binding to CD32b is about the same as comparedto wild-type homodimer. In certain embodiments the heterodimer bindingto CD32b is increased as compared to wild-type homodimer. In analternative embodiment, the heterodimer binding to CD32b is reduced ascompared to wild-type homodimer.

One of skill in the art will understand that instead of reporting theK_(D) of binding CD16a and CD32b as a ratio Fc variant to wild-typehomodimer, the K_(ID) could be reported as a ratio of Fc variant bindingto CD16a to Fc variant binding to CD32b (data not shown). This ratiowould provide an indication of the variant CH3 domain mutation on ADCC,either unchanged, increased to decreased compared to wild-type,described below in more detail.

The affinities and binding properties of the Fc variants of theinvention for an FcγR are initially determined using in vitro assays(biochemical or immunological based assays) known in the art fordetermining Fc-FcγR interactions, i.e., specific binding of an Fc regionto an FcγR including but not limited to ELISA assay, surface plasmonresonance assay, immunoprecipitation assays (See section entitled“Characterization and Functional Assays” infra) and other methods suchas indirect binding assays, competitive inhibition assays, fluorescenceresonance energy transfer (FRET), gel electrophoresis and chromatography(e.g., gel filtration). These and other methods may utilize a label onone or more of the components being examined and/or employ a variety ofdetection methods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels. A detailed description of bindingaffinities and kinetics can be found in Paul, W. E., ed., FundamentalImmunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), whichfocuses on antibody-immunogen interactions.

It is contemplated that the binding properties of the molecules of theinvention are also characterized by in vitro functional assays fordetermining one or more FcγR mediator effector cell functions (Seesection entitled “Characterization and Functional Assays” infra). Incertain embodiments, the molecules of the invention have similar bindingproperties in in viva models (such as those described and disclosedherein) as those in in vitro based assays. However, the presentinvention does not exclude molecules of the invention that do notexhibit the desired phenotype in in vitro based assays but do exhibitthe desired phenotype in vivo.

The invention encompasses Fc variants that bind FcγRIIIA (CD16a) withincreased affinity, relative to a comparable molecule. In a specificembodiment, the Fc variants of the invention bind FcγRIIIA withincreased affinity and bind FcγRIIB (CD32b) with a binding affinity thatis either unchanged or reduced, relative to a comparable molecule. Inyet another embodiment, the Fc variants of the invention have a ratio ofFcγRIIIA/FcγRIIB equilibrium dissociation constants (K_(D)) that isdecreased relative to a comparable molecule.

Also encompassed by the present invention are Fc variants that bindFcγRIIIA (CD16a) with decreased affinity, relative to a comparablemolecule. In a specific embodiment, the Fc variants of the inventionbind FcγRIIIA with decreased affinity, relative to a comparable moleculeand bind FcγRIIB with a binding affinity that is unchanged or increased,relative to a comparable molecule.

In one embodiment, the Fc variants bind with increased affinity toFcγRIIIA. In a specific embodiment, said Fc variants have affinity forFcγRIIIA that is at least 2 fold, or at least 3 fold, or at least 5fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or atleast 30 fold, or at least 40 fold, or at least 50 fold, or at least 60fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, orat least 100 fold, or at least 200 fold greater than that of acomparable molecule. In other embodiments, the Fc variants have anaffinity for FcγRIIIA that is increased by at least 10%, or at least20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%,or at least 70%, or at least S0%, or at least 90%, or at least 100%, orat least 150%, or at least 200%, relative to a comparable molecule.

In another embodiment, the Fc variant has an equilibrium dissociationconstant (K_(D)) for an Fc ligand (e.g., FcγR, C1q) that is decreasedbetween about 2 fold and 10 fold, or between about 5 fold and 50 fold,or between about 25 fold and 250 fold, or between about 100 fold and 500fold, or between about 250 fold and 1000 fold relative to a comparablemolecule.

In a another embodiment, said Fc variants have an equilibriumdissociation constant (K_(D)) for FcγRIIIA that is reduced by at least 2fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or aleast 10 fold, or at least 20 fold, or at least 30 fold, or at least 40fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, orat least 80 fold, or at least 90 fold, or at least 100 fold, or at least200 fold, or at least 400 fold, or at least 600 fold, relative to acomparable molecule. In another embodiment, the Fc variants have anequilibrium dissociation constant (K_(D)) for FcγRIIIA that is reducedby at least 10%, or at least 20%, or at least 30%, or at least 40%, orat least 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 90%, or at least 100%, or at least 150%, or at least 200%,relative to a comparable molecule.

In one embodiment, the Fc variant binds to FcγRIIB with an affinity thatis unchanged or reduced. In a specific embodiment, said Fc variants haveaffinity for FcγRIIB that is unchanged or reduced by at least 1 fold, orby at least 3 fold, or by at least 5 fold, or by at least 10 fold, or byat least 20 fold, or by at least 50 fold, or by at least 100 fold,relative to a comparable molecule. In other embodiments, the Fc variantshave an affinity for FcγRIIB that is unchanged or reduced by at least10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%,or at least 60%, or at least 70%, or at least 80%, or at least 90%, orat least 100%, or at least 150%, or at least 200%, relative to acomparable molecule.

In another embodiment, the Fc variants have an equilibrium dissociationconstant (K_(D)) for FcγRIIB that is unchanged or increased by at least2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or aleast 10 fold, or at least 20 fold, or at least 30 fold, or at least 40fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, orat least S0 fold, or at least 90 fold, or at least 100 fold, or at least200 fold relative to a comparable molecule. In another specificembodiment, the Fc variants have an equilibrium dissociation constant(K_(D)) for FcγRIIB that is unchanged or increased by at least 10%, orat least 20%, or at least 30%, or at least 40%, or at least 50%, or atleast 60%, or at least 70%, or at least 80%, or at least 90%, or atleast 100%, or at least 150%, or at least 200%, relative to a comparablemolecule.

In still another embodiment, the Fc variants bind FcγRIIIA withincreased affinity, relative to a comparable molecule and bind FcγRIIBwith an affinity that is unchanged or reduced, relative to a comparablemolecule. In a specific embodiment, the Fc variants have affinity forFcγRIIIA that is increased by at least 1 fold, or by at least 3 fold, orby at least 5 fold, or by at least 10 fold, or by at least 20 fold, orby at least 50 fold, or by at least 100 fold, relative to a comparablemolecule. In another specific embodiment, the Fc variants have affinityfor FcγRIIB that is either unchanged or is reduced by at least 2 fold,or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least10 fold, or at least 20 fold, or at least 50 fold, or at least 100 fold,relative to a comparable molecule. In other embodiments, the Fc variantshave an affinity for FcγRIIIA that is increased by at least 10%, or atleast 20%, or at least 30%, or at least 40%, or at least 50%, or atleast 60%, or at least 70%, or at least 80%, or at least 90%, or atleast 100%, or at least 150%, or at least 200%, relative to a comparablemolecule and the Fc variants have an affinity for FcγRIIB that is eitherunchanged or is increased by at least 10%, or at least 20%, or at least30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%,or at least 80%, or at least 90%, or at least 100%, or at least 150%, orat least 200%, relative to a comparable molecule.

In yet another embodiment, the Fc variants have a ratio ofFcγRIIIA/FcγRIIB equilibrium dissociation constants (K_(D)) that isdecreased relative to a comparable molecule. In a specific embodiment,the Fc variants have a ratio of FcγRIIIA/FcγRIIB equilibriumdissociation constants (K_(D)) that is decreased by at least 1 fold, orby at least 3 fold, or by at least 5 fold, or by at least 10 fold, or byat least 20 fold, or by at least 50 fold, or by at least 100 fold,relative to a comparable molecule. In another specific embodiment, theFc variants have a ratio of FcγRIIIA/FcγRIIB equilibrium dissociationconstants (K_(D)) that is decreased by at least 10%, or at least 20%, orat least 30%, or at least 40%, or at least 50%, or at least 60%, or atleast 70%, or at least 80%, or at least 90%, or at least 100%, or atleast 150%, or at least 200%, relative to a comparable molecule.

In another embodiment, the Fc variants bind FcγRIIIA with a decreasedaffinity, relative to a comparable molecule. In a specific embodiment,said Fc variants have affinity for FcγRIIIA that is reduced by at least1 fold, or by at least 3 fold, or by at least 5 fold, or by at least 10fold, or by at least 20 fold, or by at least 50 fold, or by at least 100fold, relative to a comparable molecule. In other embodiments, the Fcvariants have an affinity for FcγRIIIA that is decreased by at least10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%,or at least 60%, or at least 70%, or at least 80%, or at least 90%, orat least 100%, or at least 150%, or at least 200%, relative to acomparable molecule.

In still another embodiment, the Fc variants bind FcγRIIIA withdecreased affinity and bind FcγRIIB with an affinity that is eitherunchanged or increased, relative to a comparable molecule. In a specificembodiment, the Fc variants have affinity for FcγRIIIA that is reducedby at least 1 fold, or by at least 3 fold, or by at least 5 fold, or byat least 10 fold, or by at least 20 fold, or by at least 50 fold, or byat least 100 fold relative to a comparable molecule. In another specificembodiment, the Fc variants have affinity for FcγRIIB that is at least 2fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or aleast 10 fold, or at least 20 fold, or at least 50 fold, or at least 100fold, greater than that of a comparable molecule. In other embodiments,the Fc variants have an affinity for FcγRIIIA that is decreased by atleast 10%, or at least 20%, or at least 30%, or at least 40%, or atleast 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 90%, or at least 100%, or at least 150%, or at least 200%,relative to a comparable molecule and the Fc variants have an affinityfor FcγRIIB that is increased by at least 10%, or at least 20%, or atleast 30%, or at least 40%, or at least 50%, or at least 60%, or atleast 70%, or at least 80%, or at least 90%, or at least 100%, or atleast 150%, or at least 200%, relative to a comparable molecule.

In still another embodiment, the Fc variants have an equilibriumdissociation constant (K_(D)) for FcγRIIIA that are increased by atleast 1 fold, or by at least 3 fold, or by at least 5 fold or by atleast 10 or by at least 20 fold, or by at least 50 fold when compared tothat of a comparable molecule. In a specific embodiment, said Fcvariants have equilibrium dissociation constant (K_(D)) for FcγRIIB thatare decreased at least 2 fold, or at least 3 fold, or at least 5 fold,or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least50 fold or at least 100 fold, relative to a comparable molecule.

CH2 Variations for fcγR Selectivity

The Fc-FcγR protein-protein interaction in this complex indicates thatthe two chains in the Fc molecule interact with two distinct sites onthe FcγR molecule. Although there is symmetry in the two heavy chains inthe natural Fc molecules, the local FcγR environment around residues onone chain is different from the FcγR residues surrounding the sameresidue position on the opposite Fc chain. The two symmetry relatedpositions interact with different selection of FcγR residues.

Given the asymmetry in the association of Fc to FcγR, concurrentmutations in chain A and B of the Fc molecule do not impact theinteractions with FcγR in a symmetric manner. When introducing mutationsto optimize interactions on one chain of the Fc with its local FcγRenvironment, in a homodimeric Fc structure, the corresponding mutationin the second chain may be favorable, unfavorable or non-contributing tothe required FcγR binding and selectivity profile.

Using a structure and computation guided approach, asymmetric mutationsare engineered in the two chains of the Fc to overcome these limitationsof traditional Fc engineering strategies, which introduce the samemutations on both the chains of Fc. One can achieve better bindingselectivity between the receptors if the two chains of Fc are optimizedindependently for enhanced binding to their corresponding face of thereceptor molecule.

For instance, mutations at a particular position on one chain of the Fccan be designed to enhance selectivity to a particular residue, apositive design effort, while the same residue position can be mutatedto unfavorably interact with its local environment in an alternate Fcγreceptor type, a negative design effort, hence achieving betterselectivity between the two receptors. In certain embodiments, isprovided a method for designing asymmetric amino acid modifications inthe CH2 domain that selectively bind one Fc gamma receptor as comparedto a different Fc gamma receptor (e.g., selectively bind FcgRIIIainstead of FcgRIIb). In other certain embodiments, is provided a methodfor the design of asymmetric amino acid modifications in the CH2 domainof a variant Fc heterodimer comprising amino acid modifications in theCH3 domain to promote heterodimer formation. In another embodiment, isprovided a method to design selectivity for the different Fc gammareceptors based on a variant Fc heterodimer comprising asymmetric aminoacid modifications in the CH2 domain. In yet another embodiment, isprovided a method for designing asymmetric amino acid modifications thatbias binding of the Fc gamma receptors to one face of the Fc molecule.In other certain embodiments, is provided a method for designingpolarity drivers that bias the Fcgamma receptors to interact with onlyone face of the variant Fc heterodimer comprising asymmetric amino acidmodifications in the CH2 domain.

The asymmetric design of mutations in the CH2 domain can be tailored torecognize the FcγR on one face of the Fc molecule. This constitutes theproductive face of the asymmetric Fc scaffold while the opposite facepresents wild type like interaction propensity without the designedselectivity profile and can be considered a non-productive face. Anegative design strategy can be employed to introduce mutations on thenon-productive face to block FcγR interactions to this side of theasymmetric Fc scaffold, there by forcing the desired interactiontendencies to the Fcγ receptors.

TABLE E Potentially Interesting Selectivity Profiles of Fc for differentFcγ Receptors Receptor Binding FcγRIIIa F/V FcγRIIa H/R FcγRIIb F/YVariant ↑/— x x Selectivity x ↑/— x x x ↑/— ↑/— ↑/— x ↑/— x ↑/— x ↑/—↑/— (↑/—) indicates a variant which exhibits an increased or wild typelike binding to the particular receptor type or one of its allotype. (x)indicates no noticeable binding to the receptor or a subset allotype.

The present invention also relates to fusion polypeptides comprising abinding domain fused to an Fc region, wherein the Fc region comprising avariant CH3 domain, comprising amino acid mutations to promoteheterodimer formation with increased stability, wherein the variant CH3domain has a melting temperature (Tm) greater than 70° C. It isspecifically contemplated that molecules comprising a heterodimercomprising a variant CH3 domain may be generated by methods well knownto one skilled in the art. Briefly, such methods include but are notlimited to, combining a variable region or binding domain with thedesired specificity (e.g., a variable region isolated from a phagedisplay or expression library or derived from a human or non-humanantibody or a binding domain of a receptor) with a variant Fcheterodimers. Alternatively, one skilled in the art may generate avariant Fc heterodimer by modifying the CH3 domain in the Fc region of amolecule comprising an Fc region (e.g., an antibody).

In one embodiment, the Fc variants are antibodies or Fc fusion proteins.In a specific embodiment, the invention provides antibodies comprisingan Fc region comprising a variant CH3 domain, comprising amino acidmutations to promote heterodimer formation with increased stability,wherein the variant CH3 domain has a melting temperature (Tm) greaterthan 70° C. Such antibodies include IgG molecules that naturallycomprise an Fc region containing a CH3 domain that can be modified togenerate an Fc variant, or antibodies derivatives that have beenengineered to contain an Fc region comprising a variant CH3 domain. Fcvariants of the invention includes any antibody molecule that binds,preferably, specifically (i.e., competes off non-specific binding asdetermined by immunoassays well known in the art for assaying specificantigen-antibody binding) an antigen which comprises an Fc regionincorporating a variant CH3 domain. Such antibodies include, but are notlimited to, polyclonal, monoclonal, mono-specific, bi-specific,multi-specific, human, humanized, chimeric antibodies, single chainantibodies, Fab fragments, F(ab′)₂ fragments, disulfide-linked Fvs, andfragments containing either a VL or VH domain or even a complementarydetermining region (CDR) that specifically binds an antigen, in certaincases, engineered to contain or fused to a variant Fc heterodimer.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted antibody bound onto Fc receptors(FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK)cells, neutrophils, and macrophages) enables these cytotoxic effectorcells to bind specifically to an antigen-healing target cell andsubsequently kill the target cell with cytotoxins. Specifichigh-affinity IgG antibodies directed to the surface of target cells“arm” the cytotoxic cells and are absolutely required for such killing.Lysis of the target cell is extracellular, requires direct cell-to-cellcontact, and does not involve complement.

The ability of any particular antibody to mediate lysis of the targetcell by ADCC can be assayed. To assess ADCC activity an antibody ofinterest is added to target cells in combination with immune effectorcells, which may be activated by the antigen antibody complexesresulting in cytolysis of the target cell. Cytolysis is generallydetected by the release of label (e.g. radioactive substrates,fluorescent dyes or natural intracellular proteins) from the lysedcells. Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Specificexamples of in vitro ADCC assays are described in Wisecarver et al.,1985, 79:277; Bruggemann et al., 1987, Exp Med 166:1351; Wilkinson etal., 2001, J Immunol Methods 258:183; Patel et al., 1995 J ImmunolMethods 184:29 and herein (see section entitled “Characterization andFunctional Assays” infra). Alternatively, or additionally, ADCC activityof the antibody of interest may be assessed in vivo, e.g., in an animalmodel such as that disclosed in Clynes et al., 1998, PNAS USA 95:652.

It is contemplated that the Fc variants of the invention arecharacterized by in vitro functional assays for determining one or moreFcγR mediator effector cell functions. In specific embodiments, themolecules of the invention have similar binding properties and effectorcell functions in in vivo models (such as those described and disclosedherein) as those in in vitro based assays However, the present inventiondoes not exclude molecules of the invention that do not exhibit thedesired phenotype in in vitro based assays but do exhibit the desiredphenotype in vivo.

The present invention further provides Fc variants with enhanced CDCfunction. In one embodiment, the Fc variants have increased CDCactivity. In one embodiment, the Fc variants have CDC activity that isat least 2 fold, or at least 3 fold, or at least 5 fold, or at least 10fold, or at least 50 fold, or at least 100 fold greater than that of acomparable molecule. In another embodiment, the Fc variants bind C1qwith an affinity that is at least 2 fold, or at least 3 fold, or atleast 5 fold, or at least 7 fold, or a least 10 fold, or at least 20fold, or at least 50 fold, or at least 100 fold, greater than that of acomparable molecule. In yet another embodiment, the Fc variants have CDCactivity that is increased by at least 10%, or at least 20%, or at least30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%,or at least 80%, or at least 90%, or at least 100%, or at least 150%, orat least 200%, relative to a comparable molecule. In a specificembodiment, the Fc variants of the invention bind C1q with increasedaffinity; have enhanced CDC activity and specifically bind to at leastone antigen.

The present invention also provides Fc variants with reduced CDCfunction. In one embodiment, the Fc variants have reduced CDC activity.In one embodiment, the Fc variants have CDC activity that is at least 2fold, or at least 3 fold, or at least 5 fold or at least 10 fold or atleast 50 fold or at least 100 fold less than that of a comparablemolecule. In another embodiment, an Fc variant binds C1q with anaffinity that is reduced by at least 1 fold, or by at least 3 fold, orby at least 5 fold, or by at least 10 fold, or by at least 20 fold, orby at least 50 fold, or by at least 100 fold, relative to a comparablemolecule. In another embodiment, the Fc variants have CDC activity thatis decreased by at least 10%, or at least 20%, or at least 30%, or atleast 40%, or at least 50%, or at least 60%, or at least 70%, or atleast 80%, or at least 90%, or at least 100%, or at least 150%, or atleast 200%, relative to a comparable molecule. In a specific embodiment,Fc variants bind to C1q with decreased affinity have reduced CDCactivity and specifically bind to at least one antigen.

In some embodiments, the Fc variants comprise one or more engineeredglycoforms, i.e., a carbohydrate composition that is covalently attachedto a molecule comprising an Fc region. Engineered glycoforms may beuseful for a variety of purposes, including but not limited to enhancingor reducing effector function. Engineered glycoforms may be generated byany method known to one skilled in the art, for example by usingengineered or variant expression strains, by co-expression with one ormore enzymes, for example β(1,4)-N-acetylglucosaminyltransferase III(GnTI11), by expressing a molecule comprising an Fc region in variousorganisms or cell lines from various organisms, or by modifyingcarbohydrate(s) after the molecule comprising Fc region has beenexpressed. Methods for generating engineered glycoforms are known in theart, and include but are not limited to those described in Umana et al,1999, Nat. Biotechnol 17:176-180; Davies et al., 20017 Biotechnol Bioeng74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawaet al., 2003, J Biol Chem 278:3466-3473) U.S. Pat. No. 6,602,684; U.S.Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO01/292246A1; PCT WO 02/311140A1; PCT WO 02/30954A1; Potillegent™technology (Biowa, Inc. Princeton, N.J.); GlycoMAb™ glycosylationengineering technology (GLYCART biotechnology AG, Zurich, Switzerland).See, e.g., WO 00061739; EA01229125; US 20030115614; Okazaki et al.,2004, JMB, 336: 1239-49.

It is contemplated that Fc variants include antibodies comprising avariable region and a heterodimer Fc region, wherein the heterodimer Fcregion comprises a variant CH3 domain comprising amino acid mutations topromote heterodimer formation with increased stability, wherein thevariant CH3 domain has a melting temperature (Tm) greater than 70° C.The Fc variants which are antibodies may be produced “de novo” bycombing a variable domain, of fragment thereof, that specifically bindsat least one antigen with a heterodimer Fc region comprising a variantCH3 domain. Alternatively, heterodimer Fc variants may be produced bymodifying the CH3 domain of an Fc region containing antibody that bindsan antigen.

Antibodies of the invention may include, but are not limited to,synthetic antibodies, monoclonal antibodies, recombinantly producedantibodies, intrabodies, monospecific antibodies, multispecificantibodies, bispecific antibodies, human antibodies, humanizedantibodies, chimeric antibodies, synthetic antibodies, single-chainFvFcs (scFvFc), single-chain Fvs (scFv), and anti-idiotypic (anti-Id)antibodies. In particular, antibodies used in the methods of the presentinvention include immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules. The immunoglobulin molecules ofthe invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA andIgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass ofimmunoglobulin molecule.

Antibodies of the invention may be from any animal origin includingbirds and mammals (e.g., human, murine, donkey, sheep, rabbit, goat,guinea pig, camel, horse, or chicken). In a specific embodiment, theantibodies are human or humanized monoclonal antibodies, in particularbi-specific monoclonal antibodies. As used herein, “human” antibodiesinclude antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or from mice that express antibodies from human genes.

Antibodies like all polypeptides have an Isoelectric Point (pI), whichis generally defined as the pH at which a polypeptide carries no netcharge. It is known in the art that protein solubility is typicallylowest when the pH of the solution is equal to the isoelectric point(pI) of the protein. It is possible to optimize solubility by alteringthe number and location of ionizable residues in the antibody to adjustthe pI. For example the pI of a polypeptide can be manipulated by makingthe appropriate amino acid substitutions (e.g., by substituting acharged amino acid such as a lysine, for an uncharged residue such asalanine). Without wishing to be bound by any particular theory, aminoacid substitutions of an antibody that result in changes of the pI ofsaid antibody may improve solubility and/or the stability of theantibody. One skilled in the art would understand which amino acidsubstitutions would be most appropriate for a particular antibody toachieve a desired pI. The pI of a protein may be determined by a varietyof methods including but not limited to, isoelectric focusing andvarious computer algorithms (see for example Bjellqvist et al., 1993,Electrophoresis 14:1023). In one embodiment, the pI of the Fc variantsof the invention is between pH 6.2 and pH 8.0. In another embodiment,the pI of the antibodies of the invention is between pH 6.8 and pH 7.4.In one embodiment, substitutions resulting in alterations in the pI ofthe Fc variant of the invention will not significantly diminish itsbinding affinity for an antigen. It is contemplated that the variant CH3domain with an increased stability may also result in a change in thepI. In one embodiment, variant Fc heterodimers are specifically chosento effect both the increased stability and purity and, any desiredchange in pI.

Antibodies of the invention may be monospecific, bispecific, trispecificor have greater multispecificity. Multispecific antibodies mayspecifically bind to different epitopes of desired target molecule ormay specifically bind to both the target molecule as well as aheterologous epitope, such as a heterologous polypeptide or solidsupport material. See, e.g., International Publication Nos. WO 94/04690;WO 93/17715; WO 92/08802; WO 91/00360; and WO 92/05793; Tutt, et al.,1991, J. Immunol. 147:60-69; U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920 and 5,601,819 and Kostelny et al., 1992, J. Immuno.148:1547).

Various embodiments of multifunctional targeting molecules can bedesigned on the basis of this asymmetric scaffold as shown in FIG. 20.

Multispecific antibodies have binding specificities for at least twodifferent antigens. While such molecules normally will only bind twoantigens (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by theinstant invention. Examples of BsAbs include without limitation thosewith one arm directed against a tumor cell antigen and the other armdirected against a cytotoxic molecule, or both arms are directed againtwo different tumor cell antigens, or both arms are directed against twodifferent soluable ligands, or one arm is directed against a soluableligand and the other arm is directed against a cell surface receptor, orboth arms are directed against two different cell surface receptors.Methods for making bispecific antibodies are known in the art.

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion may bewith an immunoglobulin heavy chain constant domain, comprising at leastpart of the hinge, CH2, and CH3 regions. It is contemplated that thefirst heavy-chain constant region (CH1) containing the site necessaryfor light chain binding is present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Thisprovides for great flexibility in adjusting the mutual proportions ofthe three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. See, Example 1 and Table 2. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when, the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Antibodies with more than two valencies incorporating variant CH3domains and resulting Fc heterodimers of the invention are contemplated.For example, trispecific antibodies can be prepared. See, e.g., Tutt etal. J. Immunol. 147: 60 (1991).

Antibodies of the present invention also encompass those that havehalf-lives (e.g., serum half-lives) in a mammal, (e.g., a human), ofgreater than 15 days, greater than 20 days, greater than 25 days,greater than 30 days, greater than 35 days, greater than 40 days,greater than 45 days, greater than 2 months, greater than 3 months,greater than 4 months, or greater than 5 months. The increasedhalf-lives of the antibodies of the present invention in a mammal,(e.g., a human), results in a higher serum titer of said antibodies orantibody fragments in the mammal, and thus, reduces the frequency of theadministration of said antibodies or antibody fragments and/or reducesthe concentration of said antibodies or antibody fragments to beadministered. Antibodies having increased in vitro half-lives can begenerated by techniques known to those of skill in the art. For example,antibodies with increased in vivo half-lives can be generated bymodifying (e.g., substituting, deleting or adding) amino acid residuesidentified as involved in the interaction between the Fc domain and theFcRn receptor (see, e.g., International Publication Nos. WO 97/34631; WO04/029207; U.S. Pat. No. 6,737,056 and U.S. Patent Publication No.2003/0190311).

In a specific embodiment the variant Fc heterodimer comprising thevariant CH3 domain is a multi-specific antibody (referred to herein asan antibody of the invention), the antibody of the inventionspecifically binds an antigen of interest. In particular the antibody ofthe invention is a bi-specific antibody. In one embodiment, an antibodyof the invention specifically binds a polypeptide antigen. In anotherembodiment, an antibody of the invention specifically binds anonpolypeptide antigen. In yet another embodiment, administration of anantibody of the invention to a mammal suffering from a disease ordisorder can result in a therapeutic benefit in that mammal.

Virtually any molecule may be targeted by and/or incorporated into avariant Fc heterodimer protein (e.g., antibodies, Fc fusion proteins)including, but not limited to, the following list of proteins, as wellas subunits, domains, motifs and epitopes belonging to the followinglist of proteins: renin; a growth hormone, including human growthhormone and bovine growth hormone; growth hormone releasing factor;parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VII, factor VIIIC, factor IX, tissuefactor (TF), and von Willebrands factor; anti-clotting factors such asProtein C; atrial natriuretic factor; lung surfactant; a plasminogenactivator, such as urokinase or human urine or tissue-type plasminogenactivator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumornecrosis factor-alpha and -beta; enkephalinase; RANTES (regulated onactivation normally T-cell expressed and secreted); human macrophageinflammatory protein (MIP-1-alpha); a serum albumin such as human serumalbumin; Muellerian-inhibiting substance; relaxin A-chain; relaxinB-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbialprotein, such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyteassociated antigen (CTLA), such as CTLA-4; inhibin; activin; vascularendothelial growth factor (VEGF); receptors for hormones or growthfactors such as, for example, EGFR, VEGFR; interferons such as alphainterferon (α-IFN), beta interferon (β-IFN) and gamma interferon(γ-IFN); protein A or D; rheumatoid factors; a neurotrophic factor suchas bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or-6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor;platelet-derived growth factor (PDGF); fibroblast growth factor such asAFGF and PFGF; epidermal growth factor (EGF); transforming growth factor(TGF) such as TGF-alpha and TGF-beta, including TGF-1, TGF-2, TGF-3,TGF-4, or TGF-5; insulin-like growth factor-I and -II (IGF-I andIGF-II); des (1-3)-IGF-I (brain IGF-I), insulin-like growth factorbinding proteins; CD proteins such as CD2, CD3, CD4, CD8, CD11a, CD14,CD18, CD19, CD20, CD22, CD23, CD25, CD33, CD34, CD40, CD40L, CD52, CD63,CD64, CD80 and CD147; erythropoietin; osteoinductive factors;immunotoxins; a bone morphogenetic protein (BMP); an interferon such asinterferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs),such as M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 toIL-13; TNFα, superoxide dismutase; T-cell receptors; surface membraneproteins; decay accelerating factor; viral antigen such as, for example,a portion of the AIDS envelope, e.g., gp120; transport proteins; homingreceptors; addressins; regulatory proteins; cell adhesion molecules suchas LFA-1, Mac 1, p150.95, VLA-4, ICAM-1, ICAM-3 and VCAM, a4/p7integrin, and (Xv/p3 integrin including either a or subunits thereof,integrin alpha subunits such as CD49a, CD49b, CD49c, CD49d, CD49e,CD49f, alpha7, alpha8, alpha9, alphaD, CD11a, CD11b, CD51, CD11c, CD41,alphaIIb, alphaIELb; integrin beta subunits such as, CD29, CD 18, CD61,CD104, beta5, beta6, beta7 and beta8; Integrin subunit combinationsincluding but not limited to, αVβ3, αVβ5 and α4β7; a member of anapoptosis pathway; IgE; blood group antigens; flk2/flt3 receptor;obesity (OB) receptor; mp1 receptor; CTLA-4; protein C; an Eph receptorsuch as EphA2, EphA4, EphB2, etc.; a Human Leukocyte Antigen (HLA) suchas HLA-DR; complement proteins such as complement receptor CR1, C1Rq andother complement factors such as C3, and C5; a glycoprotein receptorsuch as GpIbα, GPIIb/IIIa and CD200; and fragments of any of theabove-listed polypeptides.

Also contemplated are antibodies of the invention that specifically bindcancer antigens including, but not limited to, ALK receptor(pleiotrophin receptor), pleiotrophin, KS 1/4 pan-carcinoma antigen;ovarian carcinoma antigen (CA125); prostatic acid phosphate; prostatespecific antigen (PSA); melanoma-associated antigen p97; melanomaantigen gp75; high molecular weight melanoma antigen (HMW-MAA); prostatespecific membrane antigen; carcinoembryonic antigen (CEA); polymorphicepithelial mucin antigen; human milk fat globule antigen; colorectaltumor-associated antigens such as: CEA, TAG-72, CO17-1A, GICA 19-9,CTA-1 and LEA; Burkitt's lymphoma antigen-38.13; CD19; human B-lymphomaantigen-CD20; CD33; melanoma specific antigens such as ganglioside GD2,ganglioside GD3, ganglioside GM2 and ganglioside GM3; tumor-specifictransplantation type cell-surface antigen (TSTA); virally-induced tumorantigens including T-antigen, DNA tumor viruses and Envelope antigens ofRNA tumor viruses; oncofetal antigen-alpha-fetoprotein such as CEA ofcolon, 514 oncofetal trophoblast glycoprotein and bladder tumoroncofetal antigen; differentiation antigen such as human lung carcinomaantigens L6 and L20; antigens of fibrosarcoma; human leukemia T cellantigen-Gp37; neoglycoprotein; sphingolipids; breast cancer antigenssuch as EGFR (Epidermal growth factor receptor); NY-BR-16; NY-BR-16 andHER2 antigen (p185HER2); polymorphic epithelial mucin (PEM); malignanthuman lymphocyte antigen-APO-1; differentiation antigen such as Iantigen found in fetal erythrocytes; primary endoderm I antigen found inadult erythrocytes; preimplantation embryos; I(Ma) found in gastricadenocarcinomas; M18, M39 found in breast epithelium; SSEA-1 found inmyeloid cells; VEP8; VEP9; Myl; Va4-D5; D₁56-22 found in colorectalcancer; TRA-1-85 (blood group H); SCP-1 found in testis and ovariancancer; C14 found in colonic adenocarcinoma; F3 found in lungadenocarcinoma; AH6 found in gastric cancer; Y hapten; Ley found inembryonal carcinoma cells; TL5 (blood group A); EGF receptor found inA431 cells; E₁ series (blood group B) found in pancreatic cancer; FC10.2found in embryonal carcinoma cells; gastric adenocarcinoma antigen;CO-514 (blood group Lea) found in Adenocarcinoma; NS-10 found inadenocarcinomas; CO-43 (blood group Leb); G49 found in EGF receptor ofA431 cells; MH2 (blood group ALeb/Ley) found in colonic adenocarcinoma;19.9 found in colon cancer; gastric cancer mucins; T₅A₇ found in myeloidcells; R₂₄ found in melanoma; 4.2, G_(D3), D1.1, OFA-1, G_(M2), OFA-2,G_(D2), and M1:22:25:8 found in embryonal carcinoma cells and SSEA-3 andSSEA-4 found in 4 to 8-cell stage embryos; Cutaneous Tcell Lymphomaantigen; MART-1 antigen; Sialy Tn (STn) antigen; Colon cancer antigenNY-CO-45; Lung cancer antigen NY-LU-12 valiant A; Adenocarcinoma antigenART1; Paraneoplastic associated brain-testis-cancer antigen(onconeuronal antigen MA2; paraneoplastic neuronal antigen);Neuro-oncological ventral antigen 2 (NOVA2); Hepatocellular carcinomaantigen gene 520; TUMOR-ASSOCIATED ANTIGEN CO-029; Tumor-associatedantigens MAGE-C1 (cancer/testis antigen CT7), MAGE-B1 (MAGE-XP antigen),MAGE-B2 (DAM6), MAGE-2, MAGE-4-a, MAGE-4-b and MAGE-X2; Cancer-TestisAntigen (NY-EOS-1) and fragments of any of the above-listedpolypeptides.

In certain embodiments, the heteromultimer described herein, comprisesat least one therapeutic antibody. In some embodiments, the therapeuticantibody binds a cancer target antigen. In an embodiment, thetherapeutic antibody may be one of is selected from the group consistingof abagovomab, adalimumab, alemtuzumab, aurograb, bapineuzumab,basiliximab, belimumab, bevacizumab, briakinumab, canakinumab,catumaxomab, certolizumab pegol, cetuximab, daclizumab, denosumab,efalizumab, galiximab, gemtuzumab ozogamicin, golimumab, ibritumomabtiuxetan, infliximab, ipilimumab, lumiliximab, mepolizumab, motavizumab,muromonab, mycograb, natalizumab, nimotuzumab, ocrelizumab, ofatumumab,omalizumab, palivizumab, panitumumab, pertuzumab, ranibizumab,reslizumab, rituximab, teplizumab, tocilizumab/atlizumab, tositumomab,trastuzumab, Proxinium™, Rencarex™, ustekinumab, zalutumumab, and anyother antibodies.

Antibodies of the invention include derivatives that are modified (i.e.,by the covalent attachment of any type of molecule to the antibody suchthat covalent attachment). For example, but not by way of limitation,the antibody derivatives include antibodies that have been modified,e.g., by glycosylation, acetylation, pegylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein,etc. Any of numerous chemical modifications may be carried out by knowntechniques, including, but not limited to, specific chemical cleavage,acetylation, formylation, metabolic synthesis of tunicamycin, etc.Additionally, the derivative may contain one or more non-classical aminoacids.

Antibodies or fragments thereof with increased in vivo half-lives can begenerated by attaching polymer molecules such as high molecular weightpolyethyleneglycol (PEG) to the antibodies or antibody fragments. PEGcan be attached to the antibodies or antibody fragments with or withouta multifunctional linker either through site-specific conjugation of thePEG to the N- or C-terminus of said antibodies or antibody fragments orvia epsilon-amino groups present on lysine residues. Linear or branchedpolymer derivatization that results in minimal loss of biologicalactivity will be used. The degree of conjugation will be closelymonitored by SDS-PAGE and mass spectrometry to ensure proper conjugationof PEG molecules to the antibodies. Unreacted PEG can be separated fromantibody-PEG conjugates by, e.g., size exclusion or ion-exchangechromatography.

Further, antibodies can be conjugated to albumin in order to make theantibody or antibody fragment more stable in vivo or have a longer halflife in vivo. The techniques are well known in the art, see e.g.,International Publication Nos. WO 93/15199, WO 93/15200, and WO01/77137; and European Patent No. EP 413,622. The present inventionencompasses the use of antibodies or fragments thereof conjugated orfused to one or more moieties, including but not limited to, peptides,polypeptides, proteins, fusion proteins, nucleic acid molecules, smallmolecules, mimetic agents, synthetic drugs, inorganic molecules, andorganic molecules.

The present invention encompasses the use of antibodies or fragmentsthereof recombinantly fused or chemically conjugated (including bothcovalent and non-covalent conjugations) to a heterologous protein orpolypeptide (or fragment thereof, for example, to a polypeptide of atleast 10, at least 20, at least 30, at least 40, at least 50, at least60, at least 70, at least 80, at least 90 or at least 100 amino acids)to generate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences. For example, antibodiesmay be used to target heterologous polypeptides to particular celltypes, either in vitro or in vivo, by fusing or conjugating theantibodies to antibodies specific for particular cell surface receptors.Antibodies fused or conjugated to heterologous polypeptides may also beused in in vitro immunoassays and purification methods using methodsknown in the art. See e.g., International publication No. WO 93/21232;European Patent No. EP 439,095; Naramura et al., 1994, Immunol. Lett.39:91-99; U.S. Pat. No. 5,474,981; Gillies et al., 1992, PNAS89:1428-1432; and Fell et al., 1991, J. Immunol. 146:2446-2452.

The present invention further includes compositions comprisingheterologous proteins, peptides or polypeptides fused or conjugated toantibody fragments. For example, the heterologous polypeptides may befused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab)₂fragment, a VH domain, a VL domain, a VH CDR, a VL CDR, or fragmentthereof. Methods for fusing or conjugating polypeptides to antibodyportions are well known in the art. See, e.g., U.S. Pat. Nos. 5,336,603;5,622,929; 5,359,046; 5,349,053; 5,447,851 and 5,112,946; EuropeanPatent Nos. EP 307,434 and EP 367,166; International publication Nos. WO96/04388 and WO 91/06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci.USA 88: 10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; andVil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341.

Additional fusion proteins, e.g. of antibodies that specifically bind anantigen (e.g., supra), may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies of the invention orfragments thereof (e.g., antibodies or fragments thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793; 5,811,238; 5,830,721; 5,834,252 and 5,837,458, and Patten etal., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, TrendsBiotechnol. 16(2): 76-82; Hansson, et al., 1999, J. Mol. Biol.287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2): 308-313.Antibodies or fragments thereof, or the encoded antibodies or fragmentsthereof, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. One or more portions of a polynucleotide encoding anantibody or antibody fragment, which portions specifically bind to anantigen may be recombined with one or more components, motifs, sections,parts, domains, fragments, etc. of one or more heterologous molecules.

The present invention further encompasses uses of variant Fcheterodimers or fragments thereof conjugated to a therapeutic agent.

An antibody or fragment thereof may be conjugated to a therapeuticmoiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, atherapeutic agent or a radioactive metal ion, e.g., alpha-emitters. Acytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include ribonuclease, monomethylauristatin E and F,paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, andcyclophosphamide and analogs or homologs thereof. Therapeutic agentsinclude, but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), and anti-mitotic agents (e.g., vincristine andvinblastine). A more extensive list of therapeutic moieties can be foundin PCT publications WO 03/075957.

Further, an antibody or fragment thereof may be conjugated to atherapeutic agent or drug moiety that modifies a given biologicalresponse. Therapeutic agents or drug moieties are not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, Onconase (or another cytotoxic RNase), pseudomonas exotoxin,cholera toxin, or diphtheria toxin; a protein such as tumor necrosisfactor, α-interferon, β-interferon, nerve growth factor, plateletderived growth factor, tissue plasminogen activator, an apoptotic agent,e.g., TNF-α, TNF-β, AIM I (see, International Publication No. WO97/33899), AIM II (see, International Publication No. WO 97/34911), FasLigand (Takahashi et al., 1994, J. Immunol., 6:1567), and VEGI (see,International Publication No. WO 99/23105), a thrombotic agent or ananti-angiogenic agent, e.g., angiostatin or endostatin; or, a biologicalresponse modifier such as, for example, a lymphokine (e.g.,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), andgranulocyte colony stimulating factor (“G-CSF”)), or a growth factor(e.g., growth hormone (“GH”)).

Moreover, an antibody can be conjugated to therapeutic moieties such asa radioactive materials or macrocyclic chelators useful for conjugatingradiometal ions (see above for examples of radioactive materials). Incertain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4:2483; Peterson et al., 1999, Bioconjug. Chem.10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943.

Methods for fusing or conjugating antibodies to polypeptide moieties areknown in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929;5,359,046; 5,349,053; 5,447,851 and 5,112,946; EP 307,434; EP 367,166;PCT Publications WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991,PNAS USA 88:10535; Zheng et al., 1995, J Immunol 154:5590; and Vil etal., 1992, PNAS USA 89:11337. The fusion of an antibody to a moiety doesnot necessarily need to be direct, but may occur through linkersequences. Such linker molecules are commonly known in the art anddescribed in Denardo et al., 1998, Clin Cancer Res 4:2483; Peterson etal., 1999, Bioconjug Chem 10:553; Zimmerman at al., 1999, Nucl Med Biol26:943; Garnett, 2002, Adv Drug Deliv Rev 53:171.

Recombinant expression of an Fc variant, derivative, analog or fragmentthereof, (e.g., an antibody or fusion protein of the invention),requires construction of an expression vector containing apolynucleotide that encodes the Fc variant (e.g., antibody, or fusionprotein). Once a polynucleotide encoding an Fc variant (e.g., antibody,or fusion protein) has been obtained, the vector for the production ofthe Fc variant (e.g., antibody, or fusion protein) may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing a protein by expressing a polynucleotidecontaining an Fc variant (e.g., antibody, or fusion protein) encodingnucleotide sequence are described herein. Methods that are well known tothose skilled in the art can be used to construct expression vectorscontaining Fc variant (e.g., antibody, or fusion protein) codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination. Theinvention, thus, provides replicable vectors comprising a nucleotidesequence encoding an Fc variant of the invention, operably linked to apromoter. Such vectors may include the nucleotide sequence encoding theconstant region of the antibody molecule (see, e.g., InternationalPublication No. WO 86/05807; International Publication No. WO 89/01036;and U.S. Pat. No. 5,122,464 and the variable domain of the antibody, ora polypeptide for generating an Fc variant may be cloned into such avector for expression of the full length antibody chain (e.g. heavy orlight chain), or complete Fc variant comprising a fusion of anon-antibody derived polypeptide and an Fc region incorporating at leastthe variant CH3 domain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an Fc variant of the invention. Thus, theinvention includes host cells containing a polynucleotide encoding an Fcvariant of the invention, operably linked to a heterologous promoter. Inspecific embodiments for the expression of Fc variants comprisingdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe Fc variants of the invention (e.g., antibody or fusion proteinmolecules) (see, e.g., U.S. Pat. No. 5,807,715). Such host-expressionsystems represent vehicles by which the coding sequences of interest maybe produced and subsequently purified, but also represent cells whichmay, when transformed or transfected with the appropriate nucleotidecoding sequences, express an Fc variant of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing Fc variantcoding sequences; yeast (e.g., Saccharomyces Pichia) transformed withrecombinant yeast expression vectors containing Fc variant codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing Fc variant codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing Fc variant coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, NSO, and 3T3 cells) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter). In certain embodiments, bacterial cells such asEscherichia coli, or eukaryotic cells, are used for the expression of anFc variant, which is a recombinant antibody or fusion protein molecules.For example, mammalian cells such as Chinese hamster ovary cells (CHO),in conjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus is an effective expressionsystem for antibodies (Foecking et al., 1986, Gene 45:101; and Cockettet al., 1990, Bio/Technology 8:2). In a specific embodiment, theexpression of nucleotide sequences encoding an Fc variant of theinvention (e.g., antibody or fusion protein) is regulated by aconstitutive promoter, inducible promoter or tissue specific promoter.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the Fcvariant (e.g., antibody or fusion protein) being expressed. For example,when a large quantity of such a protein is to be produced, for thegeneration of pharmaceutical compositions of an Fc variant, vectors thatdirect the expression of high levels of fusion protein products that arereadily purified may be desirable. Such vectors include, but are notlimited to, the E. coli expression vector pUR278 (Ruther et al., 1983,EMBO 12:1791), in which the Fc variant coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a lac Z-fusion protein is produced; pIN vectors (Inouye & Inouye,1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J.Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be usedto express foreign polypeptides as fusion proteins with glutathione5-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The Fc variant (e.g., antibody or fusionprotein) coding sequence may be cloned individually into non-essentialregions (for example the polyhedrin gene) of the virus and placed undercontrol of an AcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the Fc variant (e.g., antibody or fusion protein) codingsequence of interest may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingthe Fc variant (e.g., antibody or fusion protein) in infected hosts(e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359).Specific initiation signals may also be required for efficienttranslation of inserted antibody coding sequences. These signals includethe ATG initiation codon and adjacent sequences. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (see, e.g., Bittneret al., 1987, Methods in Enzymol. 153:516-544).

The expression of an Fc variant (e.g., antibody or fusion protein) maybe controlled by any promoter or enhancer element known in the art.Promoters which may be used to control the expression of the geneencoding an Fc variant (e.g., antibody or fusion protein) include, butare not limited to, the SV40 early promoter region (Bernoist andChambon, 1981, Nature 290:304-310), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981,Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences ofthe metallothionein gene (Brinster et al., 1982, Nature 296:39-42), thetetracycline (Tet) promoter (Gossen et al., 1995, Proc. Nat. Acad. Sci.USA 89:5547-5551); prokaryotic expression vectors such as theβ-lactamase promoter (VIIIa-Kamaroff et al, 1978, Proc. Natl. Acad. Sci.U.S.A. 75:3727-3731), or the tac promoter (DeBoer et al., 1983, Proc.Natl. Acad. Sci. U.S.A. 80:21-25; see also “Useful proteins fromrecombinant bacteria” in Scientific American, 1980, 242:74-94); plantexpression vectors comprising the nopaline synthetase promoter region(Herrera-Estrella et al., Nature 303:209-213) or the cauliflower mosaicvirus 35S RNA promoter (Gardner at al., 1981, Nucl. Acids Res. 9:2871),and the promoter of the photosynthetic enzyme ribulose biphosphatecarboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120);promoter elements from yeast or other fungi such as the Gal 4 promoter,the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)promoter, alkaline phosphatase promoter, and the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: elastase 1 gene control regionwhich is active in pancreatic acinar cells (Swift et al., 1984, Cell38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene controlregion which is active in pancreatic beta cells (Hanahan, 1985, Nature315:115-122), immunoglobulin gene control region which is active inlymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al.,1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444), mouse mammary tumor virus control region which is activein testicular, breast, lymphoid and mast cells (Leder at al., 1986, Cell45:485-495), albumin gene control region which is active in liver(Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., 1985,Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58;alpha 1-antitrypsin gene control region which is active in the liver(Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin genecontrol region which is active in myeloid cells (Mogram et al., 1985,Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basicprotein gene control region which is active in oligodendrocyte cells inthe brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286); neuronal-specific enolase (NSE) which is active inneuronal cells (Morelli et al., 1999, Gen. Virol. 80:571-83);brain-derived neurotrophic factor (BDNF) gene control region which isactive in neuronal cells (Tabuchi et al., 1998, Biochem. Biophysic. Res.Com. 253:818-823); glial fibrillary acidic protein (GFAP) promoter whichis active in astrocytes (Gomes et al., 1999, Braz J Med Biol Res 32(5):619-631; Morelli et al., 1999, Gen. Viral. 80:571-83) and gonadotropicreleasing hormone gene control region which is active in thehypothalamus (Mason at al., 1986, Science 234:1372-1378).

Expression vectors containing inserts of a gene encoding an Fc variantof the invention (e.g., antibody or fusion protein) can be identified bythree general approaches: (a) nucleic acid hybridization, (b) presenceor absence of “marker” gene functions, and (c) expression of insertedsequences. In the first approach, the presence of a gene encoding apeptide, polypeptide, protein or a fusion protein in an expressionvector can be detected by nucleic acid hybridization using probescomprising sequences that are homologous to an inserted gene encodingthe peptide, polypeptide, protein or the fusion protein, respectively.In the second approach, the recombinant vector/host system can beidentified and selected based upon the presence or absence of certain“marker” gene functions (e.g., thymidine kinase activity, resistance toantibiotics, transformation phenotype, occlusion body formation inbaculovirus, etc.) caused by the insertion of a nucleotide sequenceencoding an antibody or fusion protein in the vector. For example, ifthe nucleotide sequence encoding the Fc variant (e.g., antibody orfusion protein) is inserted within the marker gene sequence of thevector, recombinants containing the gene encoding the antibody or fusionprotein insert can be identified by the absence of the marker genefunction. In the third approach, recombinant expression vectors can beidentified by assaying the gene product (e.g., antibody or fusionprotein) expressed by the recombinant. Such assays can be based, forexample, on the physical or functional properties of the fusion proteinin in vitro assay systems, e.g., binding with anti-bioactive moleculeantibody.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered fusion protein may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, phosphorylation ofproteins). Appropriate cell lines or host systems can be chosen toensure the desired modification and processing of the foreign proteinexpressed. For example, expression in a bacterial system will produce anunglycosylated product and expression in yeast will produce aglycosylated product. Eukaryotic host cells that possess the cellularmachinery for proper processing of the primary transcript (e.g.,glycosylation, and phosphorylation) of the gene product may be used.Such mammalian host cells include, but are not limited to, CHO, VERY,BHK, Hela, COS, MDCK, 293, 3T3, W138, NSO, and in particular, neuronalcell lines such as, for example, SK-N-AS, SK-N-FI, SK-N-DZ humanneuroblastomas (Sugimoto et al., 1984, J. Natl. Cancer Inst. 73: 51-57),SK-N-SH human neuroblastoma (Biochim. Biophys. Acta, 1982, 704:450-460), Daoy human cerebellar medulloblastoma (He et al., 1992, CancerRes. 52: 1144-1148) DBTRG-05MG glioblastoma cells (Kruse et al., 1992,In Vitro Cell. Dev. Biol. 28A: 609-614), IMR-32 human neuroblastoma(Cancer Res., 1970, 30: 2110-2118), 1321N1 human astrocytoma (Proc.Natl. Acad. Sci. USA, 1977, 74: 4816), MOG-G-CCM human astrocytoma (Br.J. Cancer, 1984, 49: 269), U87MG human glioblastoma-astrocytoma (ActaPathol. Microbiol. Scand., 1968, 74: 465-486), A172 human glioblastoma(Olopade et al., 1992, Cancer Res. 52: 2523-2529), C6 rat glioma cells(Benda et al., 1968, Science 161: 370-371), Neuro-2a mouse neuroblastoma(Proc. Natl. Acad. Sci. USA, 1970, 65: 129-136), NB41A3 mouseneuroblastoma (Proc. Natl. Acad. Sci. USA, 1962, 48: 1184-1190), SCPsheep choroid plexus (Bolin et al., 1994, J. Viral. Methods 48:211-221), G355-5, PG-4 Cat normal astrocyte (Haapala et al., 1985, J.Virol. 53: 827-833), Mpf ferret brain (Trowbridge et al., 1982, In Vitro18: 952-960), and normal cell lines such as, for example, CTX TNA2 ratnormal cortex brain (Radany et al., 1992, Proc. Natl. Acad. Sci. USA 89:6467-6471) such as, for example, CRL7030 and Hs578Bst. Furthermore,different vector/host expression systems may effect processing reactionsto different extents.

For long-term, high-yield production of recombinant proteins, stableexpression is often preferred. For example, cell lines that stablyexpress an Fc variant of the invention (e.g., antibody or fusionprotein) may be engineered. Rather than using expression vectors thatcontain viral origins of replication, host cells can be transformed withDNA controlled by appropriate expression control elements (e.g.,promoter, enhancer, sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells may be allowed to growfor 1-2 days in an enriched medium, and then are switched to a selectivemedium. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci that in turn can becloned and expanded into cell lines. This method may advantageously beused to engineer cell lines that express an Fc variant that specificallybinds to an Antigen. Such engineered cell lines may be particularlyuseful in screening and evaluation of compounds that affect the activityof an Fc variant (e.g., a polypeptide or a fusion protein) thatspecifically binds to an antigen.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (Santerre et al., 1984, Gene 30:147) genes.

Once an Fc variant (e.g., antibody, or a fusion protein) of theinvention has been produced by recombinant expression, it may bepurified by any method known in the art for purification of a protein,for example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,or by any other standard technique for the purification of proteins.

The Fc variant is generally recovered from the culture medium as asecreted polypeptide, although it also may be recovered from host celllysate when directly produced without a secretory signal. If the Fcvariant is membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100).

When the Fc variant is produced in a recombinant cell other than one ofhuman origin, it is completely free of proteins or polypeptides of humanorigin. However, it is necessary to purify the Fc variant fromrecombinant cell proteins or polypeptides to obtain preparations thatare substantially homogeneous as to the Fc variant. As a first step, theculture medium or lysate is normally centrifuged to remove particulatecell debris.

Fc heterodimers having antibody constant domains can be convenientlypurified by hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography, with affinity chromatography beingthe preferred purification technique. Other techniques for proteinpurification such as fractionation on an ion-exchange column, ethanolprecipitation, reverse phase HPLC, chromatography on silica,chromatography on heparin Sepharose, chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the polypeptide to be recovered. The suitabilityof protein A as an affinity ligand depends on the species and isotype ofthe immunoglobulin Fc domain that is used. Protein A can be used topurify immunoglobulin Fc regions that are based on human γ1, γ2, or γ4heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)).Protein G is recommended for all mouse isotypes and for human γ3 (Gusset al., EMBO J. 5:15671575 (1986)). The matrix to which the affinityligand is attached is most often agarose, but other matrices areavailable. Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. The conditions forbinding an immunoadhesin to the protein A or G affinity column aredictated entirely by the characteristics of the Fc domain; that is, itsspecies and isotype. Generally, when the proper ligand is chosen,efficient binding occurs directly from unconditioned culture fluid.Bound variant Fc heterodimers can be efficiently eluted either at acidicpH (at or above 3.0), or in a neutral pH buffer containing a mildlychaotropic salt. This affinity chromatography step can result in avariant Fc heterodimer preparation that is >95% pure.

The expression levels of an Fc variant (e.g., antibody or fusionprotein) can be increased by vector amplification (for a review, seeBebbington and Hentschel, The use of vectors based on gene amplificationfor the expression of cloned genes in mammalian cells in DNA cloning,Vol. 3. (Academic Press, New York, 1987)). For example, when a marker inthe vector system expressing an antibody or fusion protein isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody or fusion protein will also increase (Crouse et al., 1983,Mol. Cell. Biol. 3:257).

The host cell may be co-transfected with two expression vectors of theinvention. For example, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors may contain identical selectable markers,which enable equal expression of heavy and light chain polypeptides.Alternatively, a single vector may be used which encodes, and is capableof expressing, a fusion protein or both heavy and light chainpolypeptides. The coding sequences for the fusion protein or heavy andlight chains may comprise cDNA or genomic DNA.

Characterization and Functional Assays

Fc variants (e.g., antibodies or fusion proteins) of the presentinvention may be characterized in a variety of ways. In one embodiment,purity of the variant Fc heterodimers is assessed using techniques wellknown in the art including, but not limited to, SDS-PAGE gels, westernblots, densitometry or mass spectrometry. Protein stability can becharacterized using an array of techniques, not limited to, sizeexclusion chromatography, UV Visible and CD spectroscopy, massspectroscopy, differential light scattering, bench top stability assay,freeze thawing coupled with other characterization techniques,differential scanning calorimetry, differential scanning fluorimetry,hydrophobic interaction chromatorgraphy, isoelectric focusing, receptorbinding assays or relative protein expression levels. In en exemplaryembodiment, stability of the variant Fc heterodimers is assessed bymelting temperature of the variant CH3 domain, as compared to wild-typeCH3 domain, using techniques well known in the art such as DifferentialScanning calorimetryor differential scanning flourimetry.

Fc variants of the present invention may also be assayed for the abilityto specifically bind to a ligand, (e.g., FcγRIIIA, FcγRIIB, C1q). Suchan assay may be performed in solution (e.g., Houghten, Bio/Techniques,13:412-421, 1992), on beads (Lam, Nature, 354:82-84, 1991, on chips(Fodor, Nature, 364:555-556, 1993), on bacteria (U.S. Pat. No.5,223,409) on plasmids (Cull et al., Proc. Natl. Acad. Sci. USA,89:1865-1869, 1992) or on phage (Scott and Smith, Science, 249:386-390,1990; Devlin, Science, 249:404-406, 1990; Cwirla et al., Proc. Natl.Acad. Sci. USA, 87:6378-6382, 1990; and Felici, J. Mol. Biol.,222:301-310, 1991). Molecules that have been identified to specificallybind to a ligand, (e.g., FcγRIIIA, FcγRIIB, C1q or to an antigen) canthen be assayed for their affinity for the ligand.

Fc variants of the invention may be assayed for specific binding to amolecule such as an antigen (e.g., cancer antigen and cross-reactivitywith other antigens) or a ligand (e.g., FcγR) by any method known in theart. Immunoassays which can be used to analyze specific binding andcross-reactivity include, but are not limited to, competitive andnon-competitive assay systems using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays, to name but a few.Such assays are routine and well known in the art (see, e.g., Ausubel etal., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York).

The binding affinity of the Fc variants of the present invention to amolecule such as an antigen or a ligand, (e.g., FcγR) and the off-rateof the interaction can be determined by competitive binding assays. Oneexample of a competitive binding assay is a radioimmunoassay comprisingthe incubation of labeled ligand, such as FcγR (e.g., 3H or 125I with amolecule of interest (e.g., Fc variants of the present invention) in thepresence of increasing amounts of unlabeled ligand, such as FcγR, andthe detection of the molecule bound to the labeled ligand. The affinityof the molecule of the present invention for the ligand and the bindingoff-rates can be determined from the saturation data by scatchardanalysis.

The kinetic parameters of an Fc variant may also be determined using anysurface plasmon resonance (SPR) based assays known in the art (e.g.,BIAcore kinetic analysis). For a review of SPR-based technology seeMullet et al., 2000, Methods 22: 77-91; Dong et al., 2002, Review inMol. Biotech., 82: 303-23; Fivash et al., 1998, Current Opinion inBiotechnology 9: 97-101; Rich et al., 2000, Current Opinion inBiotechnology 11: 54-61. Additionally, any of the SPR instruments andSPR based methods for measuring protein-protein interactions describedin U.S. Pat. Nos. 6,373,577; 6,289,286; 5,322,798; 5,341,215; 6,268,125are contemplated in the methods of the invention.

Fluorescence activated cell sorting (FACS), using any of the techniquesknown to those skilled in the art, can be used for characterizing thebinding of Fc variants to a molecule expressed on the cell surface(e.g., FcγRIIIA, FcγRIIB). Flow sorters are capable of rapidly examininga large number of individual cells that contain library inserts (e.g.,10-100 million cells per hour) (Shapiro et al., Practical Flow,Cytometry, 1995). Flow cytometers for sorting and examining biologicalcells are well known in the art. Known flow cytometers are described,for example, in U.S. Pat. Nos. 4,347,935; 5,464,581; 5,483,469;5,602,039; 5,643,796; and 6,211,477. Other known flow cytometers are theFACS Vantage™ system manufactured by Becton Dickinson and Company, andthe COPAS™ system manufactured by Union Biometrica.

The Fc variants of the invention can be characterized by their abilityto mediate FcγR-mediated effector cell function. Examples of effectorcell functions that can be assayed include, but are not limited to,antibody-dependent cell mediated cytotoxicity (ADCC), phagocytosis,opsonization, opsonophagocytosis, C1q binding, and complement dependentcell mediated cytotoxicity (CDC). Any cell-based or cell free assayknown to those skilled in the art for determining effector cell functionactivity can be used (For effector cell assays, see Perussia et al.,2000, Methods Mol. Biol. 121: 179-92; Baggiolini et al., 1998Experientia, 44(10): 841-8; Lehmann et al., 2000 J. Immunol. Methods,243(1-2): 229-42; Brown E J. 1994, Methods Cell Biol., 45: 147-64; Munnet al., 1990 J. Exp. Med., 172: 231-237, Abdul-Majid et al., 2002 Scand.J. Immunol. 55: 70-81; Ding et al., 1998, Immunity 8:403-411).

In particular, the Fc variants of the invention can be assayed forFcγR-mediated ADCC activity in effector cells, (e.g., natural killercells) using any of the standard methods known to those skilled in theart (See e.g., Perussia et al., 2000, Methods Mol. Biol. 121: 179-92).An exemplary assay for determining ADCC activity of the molecules of theinvention is based on a 51 Cr release assay comprising of: labelingtarget cells with [51 Cr]Na₂CrO₄ (this cell-membrane permeable moleculeis commonly used for labeling since it binds cytoplasmic proteins andalthough spontaneously released from the cells with slow kinetics, it isreleased massively following target cell necrosis); osponizing thetarget cells with the Fc variants of the invention; combining theopsonized radiolabeled target cells with effector cells in a microtitreplate at an appropriate ratio of target cells to effector cells;incubating the mixture of cells for 16-18 hours at 37° C.; collectingsupernatants; and analyzing radioactivity. The cytotoxicity of themolecules of the invention can then be determined, for example using thefollowing formula: % lysis=(experimental cpm−target leak cpm)/(detergentlysis cpm−target leak cpm)×100%. Alternatively, %lysis=(ADCC−AICC)/(maximum release−spontaneous release). Specific lysiscan be calculated using the formula: specific lysis=% lysis with themolecules of the invention−% lysis in the absence of the molecules ofthe invention. A graph can be generated by varying either thetarget:effector cell ratio or antibody concentration.

Method to characterize the ability of the Fc variants to bind C1q andmediate complement dependent cytotoxicity (CDC) are well known in theart. For example, to determine C1q binding, a C1q binding ELISA may beperformed. An exemplary assay may comprise the following: assay platesmay be coated overnight at 4 C with polypeptide variant or startingpolypeptide (control) in coating buffer. The plates may then be washedand blocked. Following washing, an aliquot of human C1q may be added toeach well and incubated for 2 hrs at room temperature. Following afurther wash, 100 uL of a sheep anti-complement C1q peroxidaseconjugated antibody may be added to each well and incubated for 1 hourat room temperature. The plate may again be washed with wash buffer and100 ul of substrate buffer containing OPD (O-phenylenediaminedihydrochloride (Sigma)) may be added to each well. The oxidationreaction, observed by the appearance of a yellow color, may be allowedto proceed for 30 minutes and stopped by the addition of 100 ul of 4.5NH2SO4. The absorbance may then read at (492-405) nm.

To assess complement activation, a complement dependent cytotoxicity(CDC) assay may be performed, (e.g. as described in Gazzano-Santoro etal., 1996, J. Immunol. Methods 202:163). Briefly, various concentrationsof Fc variant and human complement may be diluted with buffer. Cellswhich express the antigen to which the Fc variant binds may be dilutedto a density of about 1×106 cells/ml. Mixtures of the Fc variant,diluted human complement and cells expressing the antigen may be addedto a flat bottom tissue culture 96 well plate and allowed to incubatefor 2 hrs at 37 C. and 5% CO2 to facilitate complement mediated celllysis. 50 uL of alamar blue (Accumed International) may then be added toeach well and incubated overnight at 37 C. The absorbance is measuredusing a 96-well fluorometer with excitation at 530 nm n and emission at590 nm. The results may be expressed in relative fluorescence units(RFU). The sample concentrations may be computed from a standard curveand the percent activity, relative to a comparable molecule (i.e., amolecule comprising an Fc region with an unmodified or wild type CH3domain) is reported for the Fc variant of interest.

Complement assays may be performed with guinea pig, rabbit or humanserum. Complement lysis of target cells may be detected by monitoringthe release of intracellular enzymes such as lactate dehydrogenase(LDH), as described in Korzeniewski et al., 1983, Immunol. Methods64(3): 313-20; and Decker et al., 1988, J. Immunol. Methods 115(1):61-9; or the release of an intracellular label such as europium,chromium 51 or indium 111 in which target cells are labeled.

Methods

The present invention encompasses administering one or more Fc variantof the invention (e.g., antibodies) to an animal, in particular amammal, specifically, a human, for preventing, treating, or amelioratingone or more symptoms associated with a disease, disorder, or infection.The Fc variants of the invention are particularly useful for thetreatment or prevention of a disease or disorder where an alteredefficacy of effector cell function (e.g., ADCC, CDC) is desired. The Fcvariants and compositions thereof are particularly useful for thetreatment or prevention of primary or metastatic neoplastic disease(i.e., cancer), and infectious diseases. Molecules of the invention maybe provided in pharmaceutically acceptable compositions as known in theart or as described herein. As detailed below, the molecules of theinvention can be used in methods of treating or preventing cancer(particularly in passive immunotherapy), autoimmune disease,inflammatory disorders or infectious diseases.

The Fc variants of the invention may also be advantageously utilized incombination with other therapeutic agents known in the art for thetreatment or prevention of a cancer, autoimmune disease, inflammatorydisorders or infectious diseases. In a specific embodiment, Fc variantsof the invention may be used in combination with monoclonal or chimericantibodies, lymphokines, or hematopoietic growth factors (such as, e.g.,IL-2, IL-3 and IL-7), which, for example, serve to increase the numberor activity of effector cells which interact with the molecules and,increase immune response. The Fc variants of the invention may also beadvantageously utilized in combination with one or more drugs used totreat a disease, disorder, or infection such as, for example anti-canceragents, anti-inflammatory agents or anti-viral agents.

Accordingly, the present invention provides methods for preventing,treating, or ameliorating one or more symptoms associated with cancerand related conditions by administering one or more Fc variants of theinvention. Although not intending to be bound by any mechanism ofactions, an Fc variant of the invention that binds FcγRIIIA and/orFcγRIIA with a greater affinity than a comparable molecule, and furtherbinds FcγRIIB with a lower affinity than a comparable molecule, and/orsaid Fc variant has an enhanced effector function, e.g., ADCC, CDC,phagocytosis, opsonization, etc. will result in the selective targetingand efficient destruction of cancer cells.

The invention further encompasses administering one or more Fc variantsof the invention in combination with other therapies known to thoseskilled in the art for the treatment or prevention of cancer, includingbut not limited to, current standard and experimental chemotherapies,hormonal therapies, biological therapies, immunotherapies, radiationtherapies, or surgery. In some embodiments, the molecules of theinvention may be administered in combination with a therapeutically orprophylactically effective amount of one or more anti-cancer agents,therapeutic antibodies or other agents known to those skilled in the artfor the treatment and/or prevention of cancer. Examples of dosingregimes and therapies which can be used in combination with the Fcvariants of the invention are well known in the art and have beendescribed in detail elsewhere (see for example, PCT publications WO02/070007 and WO 03/075957).

Cancers and related disorders that can be treated or prevented bymethods and compositions of the present invention include, but are notlimited to, the following: Leukemias, lymphomas, multiple myelomas, boneand connective tissue sarcomas, brain tumors, breast cancer, adrenalcancer, thyroid cancer, pancreatic cancer, pituitary cancers, eyecancers, vaginal cancers, vulvar cancer, cervical cancers, uterinecancers, ovarian cancers, esophageal cancers, stomach cancers, coloncancers, rectal cancers, liver cancers, gallbladder cancers,cholangiocarcinomas, lung cancers, testicular cancers, prostate cancers,penal cancers; oral cancers, salivary gland cancers pharynx cancers,skin cancers, kidney cancers, bladder cancers (for a review of suchdisorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. LippincottCo., Philadelphia and Murphy et al., 1997, Informed Decisions: TheComplete Book of Cancer Diagnosis, Treatment, and Recovery, VikingPenguin, Penguin Books U.S.A., Inc., United States of America).

The invention further contemplates engineering any of the antibodiesknown in the art for the treatment and/or prevention of cancer andrelated disorders, so that the antibodies comprise an Fc regionincorporating a variant CH3 domain of the invention.

In a specific embodiment, a molecule of the invention (e.g., an antibodycomprising a variant Fc heterodimer inhibits or reduces the growth ofprimary tumor or metastasis of cancerous cells by at least 99%, at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, at least 50%, at least 45%, at least 40%, at least45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least10% relative to the growth of primary tumor or metastasis in the absenceof said molecule of the invention.

The present invention encompasses the use of one or more Fc variants ofthe invention for preventing, treating, or managing one or more symptomsassociated with an inflammatory disorder in a subject. Although notintending to be bound by any mechanism of actions, Fc variants withenhanced affinity for FcγRIIB will lead to a dampening of the activatingreceptors and thus a dampening of the immune response and havetherapeutic efficacy for treating and/or preventing an autoimmunedisorder. Furthermore, antibodies binding more than one target, such asbispecific antibodies comprising a variant Fc heterodimer, associatedwith an inflammatory disorder may provide synergist effects overmonovalent therapy.

The invention further encompasses administering the Fc variants of theinvention in combination with a therapeutically or prophylacticallyeffective amount of one or more anti-inflammatory agents. The inventionalso provides methods for preventing, treating, or managing one or moresymptoms associated with an autoimmune disease further comprising,administering to said subject an Fc variant of the invention incombination with a therapeutically or prophylactically effective amountof one or more immunomodulatory agents. Examples of autoimmune disordersthat may be treated by administering the Fc variants of the inventioninclude, but are not limited to, alopecia greata, ankylosingspondylitis, antiphospholipid syndrome, autoimmune Addison's disease,autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmunethrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy,celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome(CFIDS), chronic inflammatory demyelinating polyneuropathy,Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, coldagglutinin disease, Crohn's disease, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis,Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathicpulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgAneuropathy, juvenile arthritis, lichen planus, lupus erthematosus,Meniere's disease, mixed connective tissue disease, multiple sclerosis,type 1 or immune-mediated diabetes mellitus, myasthenia gravis,pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,polychrondritis, polyglandular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobulinemia, primarybiliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld'sphenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis,scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupuserythematosus, lupus erythematosus, takayasu arteritis, temporalarteristis/giant cell arteritis, ulcerative colitis, uveitis,vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, andWegener's granulomatosis. Examples of inflamatory disorders include, butare not limited to, asthma, encephilitis, inflammatory bowel disease,chronic obstructive pulmonary disease (COPD), allergic disorders, septicshock, pulmonary fibrosis, undifferentiated spondyloarthropathy,undifferentiated arthropathy, arthritis, inflammatory osteolysis, andchronic inflammation resulting from chronic viral or bacteriainfections. Some autoimmune disorders are associated with aninflammatory condition, thus, there is overlap between what isconsidered an autoimmune disorder and an inflammatory disorder.Therefore, some autoimmune disorders may also be characterized asinflammatory disorders. Examples of inflammatory disorders which can beprevented, treated or managed in accordance with the methods of theinvention include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentiated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections.

Fc variants of the invention can also be used to reduce the inflammationexperienced by animals, particularly mammals, with inflammatorydisorders. In a specific embodiment, an Fc of the invention reduces theinflammation in an animal by at least 99%, at least 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, atleast 50%, at least 45%, at least 40%, at least 45%, at least 35%, atleast 30%, at least 25%, at least 20%, or at least 10% relative to theinflammation in an animal, which is not administered the said molecule.

The invention further contemplates engineering any of the antibodiesknown in the art for the treatment and/or prevention of autoimmunedisease or inflammatory disease, so that the antibodies comprise avariant Fc heterodimer of the invention.

The invention also encompasses methods for treating or preventing aninfectious disease in a subject comprising administering atherapeutically or prophylactically effective amount of one or more Fcvariants of the invention. Infectious diseases that can be treated orprevented by the Fc variants of the invention are caused by infectiousagents including but not limited to viruses, bacteria, fungi, protozae,and viruses.

Viral diseases that can be treated or prevented using the Fc variants ofthe invention in conjunction with the methods of the present inventioninclude, but are not limited to, those caused by hepatitis type A,hepatitis type B, hepatitis type C, influenza, varicella, adenovirus,herpes simplex type I (HSV-I), herpes simplex type II (HSV-II),rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytialvirus, papilloma virus, papova virus, cytomegalovirus, echinovirus,arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus,rubella virus, polio virus, small pox, Epstein Barr virus, humanimmunodeficiency virus type I (HIV-I), human immunodeficiency virus typeII (HIV-II), and agents of viral diseases such as viral meningitis,encephalitis, dengue or small pox.

Bacterial diseases that can be treated or prevented using the Fcvariants of the invention in conjunction with the methods of the presentinvention, that are caused by bacteria include, but are not limited to,mycobacteria rickettsia, mycoplasma, neisseria, S. pneumonia, Borreliaburgdorferi (Lyme disease), Bacillus antracis (anthrax), tetanus,streptococcus, staphylococcus, mycobacterium, tetanus, pertissus,cholera, plague, diptheria, chlamydia, S. aureus and legionella.Protozoal diseases that can be treated or prevented using the moleculesof the invention in conjunction with the methods of the presentinvention, that are caused by protozoa include, but are not limited to,leishmania, kokzidioa, trypanosoma or malaria. Parasitic diseases thatcan be treated or prevented using the molecules of the invention inconjunction with the methods of the present invention, that are causedby parasites include, but are not limited to, chlamydia and rickettsia.

In some embodiments, the Fc variants of the invention may beadministered in combination with a therapeutically or prophylacticallyeffective amount of one or additional therapeutic agents known to thoseskilled in the art for the treatment and/or prevention of an infectiousdisease. The invention contemplates the use of the molecules of theinvention in combination with other molecules known to those skilled inthe art for the treatment and or prevention of an infectious diseaseincluding, but not limited to, antibiotics, antifungal agents andanti-viral agents.

The invention provides methods and pharmaceutical compositionscomprising Fc variants of the invention (e.g., antibodies,polypeptides). The invention also provides methods of treatment,prophylaxis, and amelioration of one or more symptoms associated with adisease, disorder or infection by administering to a subject aneffective amount of at least one Fc variant of the invention, or apharmaceutical composition comprising at least one Fc variant of theinvention. In a one aspect, the Fc variant, is substantially purified(i.e., substantially free from substances that limit its effect orproduce undesired side-effects this includes homodimers and othercellular material). In a specific embodiment, the subject is an animal,such as a mammal including non-primates (e.g., cows, pigs, horses, cats,dogs, rats etc.) and primates (e.g., monkey such as, a cynomolgousmonkey and a human). In a specific embodiment, the subject is a human.In yet another specific embodiment, the antibody of the invention isfrom the same species as the subject.

The route of administration of the composition depends on the conditionto be treated. For example, intravenous injection may be preferred fortreatment of a systemic disorder such as a lymphatic cancer or a tumorthat has metastasized. The dosage of the compositions to be administeredcan be determined by the skilled artisan without undue experimentationin conjunction with standard dose-response studies. Relevantcircumstances to be considered in making those determinations includethe condition or conditions to be treated, the choice of composition tobe administered, the age, weight, and response of the individualpatient, and the severity of the patient's symptoms. Depending on thecondition, the composition can be administered orally, parenterally,intranasally, vaginally, rectally, lingually, sublingually, buccally,intrabuccally and/or transdermally to the patient.

Accordingly, compositions designed for oral, lingual, sublingual, buccaland intrabuccal administration can be made without undue experimentationby means well known in the art, for example, with an inert diluent orwith an edible carrier. The composition may be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the pharmaceutical compositions of the present inventionmay be incorporated with excipients and used in the form of tablets,troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums,and the like.

Tablets, pills, capsules, troches and the like may also contain binders,recipients, disintegrating agent, lubricants, sweetening agents, and/orflavoring agents. Some examples of binders include microcrystallinecellulose, gum tragacanth and gelatin. Examples of excipients includestarch and lactose. Some examples of disintegrating agents includealginic acid, cornstarch, and the like. Examples of lubricants includemagnesium stearate and potassium stearate. An example of a glidant iscolloidal silicon dioxide. Some examples of sweetening agents includesucrose, saccharin, and the like. Examples of flavoring agents includepeppermint, methyl salicylate, orange flavoring, and the like. Materialsused in preparing these various compositions should be pharmaceuticallypure and non-toxic in the amounts used.

The pharmaceutical compositions of the present invention can beadministered parenterally, such as, for example, by intravenous,intramuscular, intrathecal and/or subcutaneous injection. Parenteraladministration can be accomplished by incorporating the compositions ofthe present invention into a solution or suspension. Such solutions orsuspensions may also include sterile diluents, such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol and/or other synthetic solvents. Parenteralformulations may also include antibacterial agents, such as, forexample, benzyl alcohol and/or methyl parabens, antioxidants, such as,for example, ascorbic acid and/or sodium bisulfite, and chelatingagents, such as EDTA. Buffers, such as acetates, citrates andphosphates, and agents for the adjustment of tonicity, such as sodiumchloride and dextrose, may also be added. The parenteral preparation canbe enclosed in ampules, disposable syringes and/or multiple dose vialsmade of glass or plastic. Rectal administration includes administeringthe composition into the rectum and/or large intestine. This can beaccomplished using suppositories and/or enemas. Suppository formulationscan be made by methods known in the art. Transdermal administrationincludes percutaneous absorption of the composition through the skin.Transdermal formulations include patches, ointments, creams, gels,salves, and the like. The compositions of the present invention can beadministered nasally to a patient. As used herein, nasally administeringor nasal administration includes administering the compositions to themucous membranes of the nasal passage and/or nasal cavity of thepatient.

The pharmaceutical compositions of the invention may be used inaccordance with the methods of the invention for preventing, treating,or ameliorating one or more symptoms associated with a disease,disorder, or infection. It is contemplated that the pharmaceuticalcompositions of the invention are sterile and in suitable form foradministration to a subject.

In one embodiment the compositions of the invention are pyrogen-freeformulations that are substantially free of endotoxins and/or relatedpyrogenic substances. Endotoxins include toxins that are confined insidea microorganism and are released when the microorganisms are broken downor die. Pyrogenic substances also include fever-inducing, thermostablesubstances (glycoproteins) from the outer membrane of bacteria and othermicroorganisms. Both of these substances can cause fever, hypotensionand shock if administered to humans. Due to the potential harmfuleffects, it is advantageous to remove even low amounts of endotoxinsfrom intravenously administered pharmaceutical drug solutions. The Food& Drug Administration (“FDA”) has set an upper limit of 5 endotoxinunits (EU) per dose per kilogram body weight in a single one hour periodfor intravenous drug applications (The United States PharmacopeialConvention, Pharmacopeial Forum 26 (1):223 (2000)). When therapeuticproteins are administered in amounts of several hundred or thousandmilligrams per kilogram body weight, as can be the case with monoclonalantibodies, it is advantageous to remove even trace amounts ofendotoxin. In a specific embodiment, endotoxin and pyrogen levels in thecomposition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then0.001 EU/mg.

The invention provides methods for preventing, treating, or amelioratingone or more symptoms associated with a disease, disorder, or infection,said method comprising: (a) administering to a subject in need thereof adose of a prophylactically or therapeutically effective amount of acomposition comprising one or more Fc variants and (b) administering oneor more subsequent doses of said Fc variants, to maintain a plasmaconcentration of the Fc variant at a desirable level (e.g., about 0.1 toabout 100 μg/ml), which continuously binds to an antigen. In a specificembodiment, the plasma concentration of the Fc variant is maintained at10 μg/ml, 15 μg/ml, 20 μg/ml, 25 μg/ml, 30 μg/ml, 35 μg/ml, 40 μg/ml, 45μg/ml or 50 μg/ml. In a specific embodiment, said effective amount of Fcvariant to be administered is between at least 1 mg/kg and 8 mg/kg perdose. In another specific embodiment, said effective amount of Fcvariant to be administered is between at least 4 mg/kg and 5 mg/kg perdose. In yet another specific embodiment, said effective amount of Fcvariant to be administered is between 50 mg and 250 mg per dose. Instill another specific embodiment, said effective amount of Fc valiantto be administered is between 100 mg and 200 mg per dose.

The present invention also encompasses protocols for preventing,treating, or ameliorating one or more symptoms associated with adisease, disorder, or infection which an Fc variant is used incombination with a therapy (e.g., prophylactic or therapeutic agent)other than an Fc variant and/or variant fusion protein. The invention isbased, in part, on the recognition that the Fc variants of the inventionpotentiate and synergize with, enhance the effectiveness of, improve thetolerance of, and/or reduce the side effects caused by, other cancertherapies, including current standard and experimental chemotherapies.The combination therapies of the invention have additive potency, anadditive therapeutic effect or a synergistic effect. The combinationtherapies of the invention enable lower dosages of the therapy (e.g.,prophylactic or therapeutic agents) utilized in conjunction with Fcvariants for preventing, treating, or ameliorating one or more symptomsassociated with a disease, disorder, or infection and/or less frequentadministration of such prophylactic or therapeutic agents to a subjectwith a disease disorder, or infection to improve the quality of life ofsaid subject and/or to achieve a prophylactic or therapeutic effect.Further, the combination therapies of the invention reduce or avoidunwanted or adverse side effects associated with the administration ofcurrent single agent therapies and/or existing combination therapies,which in turn improves patient compliance with the treatment protocol.Numerous molecules which can be utilized in combination with the Fcvariants of the invention are well known in the art. See for example,PCT publications WO 02/070007; WO 03/075957 and U.S. Patent Publication2005/064514.

The present invention provides kits comprising one or more Fc variantswith altered binding affinity to FcγRs and/or C1q and altered ADCCand/or CDC activity that specifically bind to an antigen conjugated orfused to a detectable agent, therapeutic agent or drug, in one or morecontainers, for use in monitoring, diagnosis, preventing, treating, orameliorating one or more symptoms associated with a disease, disorder,or infection.

EXAMPLES

The examples below are given so as to illustrate the practice of thisinvention. They are not intended to limit or define the entire scope ofthis invention.

Example 1 Generation of Bivalent Monospecific Antibodies withHeterodimer Fc Domains

The genes encoding the antibody heavy and light chains were constructedvia gene synthesis using codons optimized for human/mammalianexpression. The Fab sequences were generated from a known Her2/neubinding Ab (Carter P. et al. (1992) Humanization of an anti P185 Her2antibody for human cancer therapy. Proc Natl Acad Sci 89, 4285.) and theFc was an IgG1 isotype (SEQ ID NO:1). The final gene products weresub-cloned into the mammalian expression vector pTT5 (NRC-BRI, Canada)(Durocher, Y., Perret, S. & Kamen, A. High-level and high-throughputrecombinant protein production by transient transfection ofsuspension-growing human HEK293-EBNA1 cells. Nucleic acids research 30,E9 (2002)). The mutations in the CH3 domain were introduced viasite-directed mutagenesis of the pTT5 template vectors. See Table 1 andTable 6 and Table 7 for a list of the variant CH3 domain mutations made.

In order to estimate the formation of heterodimers and determine theratio of homodimers vs. heterodimers the two heterodimer heavy chainswere designed with C-terminal extensions of different size(specifically, chain A with C-terminal HisTag and chain B withC-terminal mRFP plus StrepTagII). This difference in molecular weightallows differentiation of homodimers vs. heterodimer in non-reducingSDS-PAGE as illustrated in FIG. 25A.

The HEK293 cells were transfected in exponential growth phase (1.5 to 2million cells/mL) with aqueous 1 mg/mL 25 kDa polyethylenimine (PEI,Polysciences) at a PEI:DNA ratio of 2.5:1 (Raymond C. et al. Asimplified polyethylenimine-mediated transfection process forlarge-scale and high-throughput applications. Methods. 55(1):44-51(2011)). In order to determine the optimal concentration range forforming heterodimers, the DNA was transfected in three separate ratiosof the two heavy chains. For example, this was done in 2 ml culturevolume and transfection DNA, comprised of 5% GFP, 45% salmon sperm DNA,25% light chain and 25% total heavy chains, where the heavy chain Aplasmid (with C-terminal His-Tag) and the heavy chain B plasmid (withC-terminal StrepTagII plus RFP) at 65%/55%/35% or 10%/20%/40%) weresampled at 3 different relative ratios (chain_A(His)/chain_B(mRFP)) of10%/65%; 20%/55%; 40%/35% (the apparent 1:1 expression ratio of aWT_His/WT_mRFP heterodimer was determined to be close to the DNA ratio20%/55%). At 4 to 48 hours after transfection in F17 serum-free media(Gibco), TN1 peptone is added to a final concentration of 0.5%.Expressed antibody was analyzed by SDS-PAGE to determine the best ratioof heavy to light chain for optimal heterodimer formation (See FIGS. 25Band C).

A selected DNA ratio, for example 50% light chain plasmid, 25% heavychain A plasmid, 25% heavy chain B of AZ33 and AZ34, with 5% GFP, and45% salmon sperm DNA was used to transfect 150 mL of cell culture asdescribed above. Transfected cells were harvested after 5-6 days withthe culture medium collected after centrifugation at 4000 rpm andclarified using a 0.45 μm filter. See Table 2 below, for a list of thepercentage of light and heavy chain A and B plasmids used in the scaleup transfection assays for each of the antibodies with CH3 mutationsgenerated for further analysis, including determination of purity andmelting temperature.

TABLE 2 Variant LC/HCA/HCB Wild-Type 50%, 50% AZ12 50%, 25%, 25% AZ1450%, 25%, 25% AZ15 50%, 25%, 25% AZ17 50%, 25%, 25% AZ19 50%, 25%, 25%AZ20 50%, 25%, 25% AZ21 50%, 25%, 25% AZ25 50%, 25%, 25% AZ29 50%, 25%,25% AZ30 50%, 25%, 25% AZ32 50%, 25%, 25% AZ33 50%, 25%, 25% AZ34 50%,25%, 25% AZ42 50%, 25%, 25% AZ44 50%, 25%, 25% AZ46 50%, 25%, 25% AZ4750%, 25%, 25% AZ48 40%, 25%, 35% AZ49 50%, 25%, 25% AZ63 50%, 20%, 30%AZ64 50%, 20%, 30% AZ65 50%, 20%, 30% AZ66 50%, 20%, 30% AZ67 50%, 20%,30% AZ68 50%, 20%, 30% AZ69 50%, 20%, 30% AZ70 50%, 20%, 30% AZ71 40%,20%, 40% AZ72 40%, 20%, 40% AZ73 40%, 20%, 40% AZ74 40%, 20%, 40% AZ7540%, 20%, 40% AZ76 40%, 20%, 40% AZ77 40%, 20%, 40% AZ78 50%, 20%, 30%AZ79 25%, 35%, 40% AZ81 25%, 35%, 40% AZ82 50%, 20%, 30% AZ83 50%, 20%,30% AZ84 50%, 20%, 30% AZ85 50%, 25%, 25% AZ86 40%, 15%, 45% AZ87 50%,25%, 25% AZ88 50%, 25%, 25% AZ89 40%, 15%, 45% AZ91 50%, 25%, 25% AZ9240%, 20%, 40% AZ93 40%, 20%, 40% AZ94 50%, 25%, 25% AZ95 50%, 20%, 30%AZ98 50%, 20%, 30% AZ100 50%, 20%, 30% AZ101 50%, 20%, 30% AZ106 25%,35%, 40% AZ114 25%, 20%, 55% AZ115 25%, 20%, 55% AZ122 25%, 20%, 55%AZ123 40%, 20%, 40% AZ124 40%, 20%, 40% AZ129 40%, 30%, 30% AZ130 40%,30%, 30%

Example 2 Purification of Bivalent Monospecific Antibodies withHeterodimer Fc Domains

The clarified culture medium was loaded onto a MabSelect SuRe (GEHealthcare) protein-A column and washed with 10 column volumes of PBSbuffer at pH 7.2. The antibody was eluted with 10 column volumes ofcitrate buffer at pH 3.6 with the pooled fractions containing theantibody neutralized with TRIS at pH 11. The protein was finallydesalted using an Econo-Pac 10DG column (Bio-Rad). The C-terminal mRFPtag on the heavy chain B was removed by incubating the antibody withenterokinase (NEB) at a ratio of 1:10,000 overnight in PBS at 25° C. Theantibody was purified from the mixture by gel filtration. For gelfiltration, 3.5 mg of the antibody mixture was concentrated to 1.5 mLand loaded onto a Sephadex 200 HiLoad 16/600 200 pg column (GEHealthcare) via an AKTA Express FPLC at a flow-rate of 1 mL/min. PBSbuffer at pH 7.4 was used at a flow-rate of 1 mL/min. Fractionscorresponding to the purified antibody were collected, concentrated to˜1 mg/mL and stored at −80° C.

Formation of heterodimers, as compared to homodimers, was assayed usingnon-reducing SDS-PAGE and mass spectrometry. Protein A purified antibodywas run on a 4-12% gradient SDS-PAGE, non-reducing gel to determine thepercentage of heterodimers formed prior to enterokinase (EK) treatment(See, FIG. 26). For mass spectrometry, all Trap LC/MS (ESI-TOF)experiments were performed on an Agilent 1100 HPLC system interfacedwith a Waters Q-TOF2 mass spectrometer. Five μg of gel filtrationpurified antibody was injected into a Protein MicroTrap (1.0 by 8.0 mm),washed with 1% acetonitrile for 8 minutes, a gradient from 1 to 20%acetonitrile/0.1% formic acid for 2 minutes, then eluted with a 20 to60% acetonitrile/0.1% formic acid gradient for 20 minutes. Eluate (30-50μL/min) was directed to the spectrometer with spectrum acquired everysecond (m/z 800 to 4,000). (See, FIG. 28) Variants having greater than90% heterodimers were selected for further analysis, with the exceptionof AZ12 and AZ14 which each had greater than 85% heterodimer formation.

Example 3 Stability Determination of Bivalent Monospecific Antibodieswith Heterodimer Fc Domains Using Differential Scanning Calorimetry(DSC)

All DSC experiments were carried out using a GE VP-Capillary instrument.The proteins were buffer-exchanged into PBS (pH 7.4) and diluted to 0.4to 0.5 mg/mL with 0.137 mL loaded into the sample cell and measured witha scan rate of 1° C./min from 20 to 100° C. Data was analyzed using theOrigin software (GE Healthcare) with the PBS buffer backgroundsubtracted. (See, FIG. 27). See Table 3 for a list of variants testedand a melting temperature determined. See Table 4 for a list of thevariants with a melting temperature of 70° C. and above and the specificTm for each variant.

TABLE 3 Melting temperature measurements of variant CH3 domains in anIgG1 antibody having 90% or more heterodimer formation compared tohomodimer formation Variant Tm ° C. Wild-Type 81 Control 1 69 Control 269 AZ3 65 AZ6 68 AZ8 68 AZ12 77 AZ14 77 AZ15 71.5 AZ16 68.5 AZ17 71 AZ1869.5 AZ19 70.5 AZ20 70 AZ21 70 AZ22 69 AZ23 69 AZ24 69.5 AZ25 70.5 AZ2669 AZ27 68 AZ28 69.5 AZ29 70 AZ30 71 AZ31 68 AZ32 71.5 AZ33 74 AZ34 73.5AZ38 69 AZ42 70 AZ43 67 AZ44 71.5 AZ46 70.5 AZ47 70.5 AZ48 70.5 AZ49 71AZ50 69 AZ52 68 AZ53 68 AZ54 67 AZ58 69 AZ59 69 AZ60 67 AZ61 69 AZ62 68AZ63 71.5 AZ64 74 AZ65 73 AZ66 72.5 AZ67 72 AZ68 72 AZ69 71 AZ70 75.5AZ71 71 AZ72 70.5 AZ73 71 AZ74 71 AZ75 70 AZ76 71.5 AZ77 71 AZ78 70 AZ7970 AZ81 70.5 AZ82 71 AZ83 71 AZ84 71.5 AZ85 71.5 AZ86 72.5 AZ87 71 AZ8872 AZ89 72.5 AZ91 71.5 AZ92 71.5 AZ93 71.5 AZ94 73.5 AZ95 72 AZ98 70AZ99 69 AZ100 71.5 AZ101 74 AZ106 74 AZ114 71 AZ115 70 AZ117 69.5 AZ12271 AZ123 70 AZ124 70 AZ125 69 AZ126 69 AZ129 70.5 AZ130 71

TABLE 4 Melting temperature measurements of select variant CH3 domainsin an IgG1 antibody Variant Tm ° C. Wild-Type 81.5 Control 1 69 Control2 69 AZ12 >77 AZ14 >77 AZ15 71.5 AZ17 71 AZ19 70.5 AZ20 70 AZ21 70 AZ2570.5 AZ29 70 AZ30 71 AZ32 71.5 AZ33 74 AZ34 73.5 AZ42 70 AZ44 71.5 AZ4670.5 AZ47 70.5 AZ48 70.5 AZ49 71 AZ63 71.5 AZ64 74 AZ65 73 AZ66 72.5AZ67 72 AZ68 72 AZ69 71 AZ70 75.5 AZ71 71 AZ72 70.5 AZ73 71 AZ74 71 AZ7570 AZ76 71.5 AZ77 71 AZ78 70 AZ79 70 AZ81 70.5 AZ82 71 AZ83 71 AZ84 71.5AZ85 71.5 AZ86 72.5 AZ87 71 AZ88 72 AZ89 72.5 AZ91 71.5 AZ92 71.5 AZ9371.5 AZ94 73.5 AZ95 72 AZ98 70 AZ100 71.5 AZ101 74 AZ106 74 AZ114 71AZ115 70 AZ122 71 AZ123 70 AZ124 70 AZ129 70.5 AZ130 71

Example 4 Evaluation of FcgammaR Binding Using Surface Plasmon Resonance

All binding experiments were carried out using a BioRad ProteOn XPR36instrument at 25° C. with 10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and0.05% Tween 20 at pH 7.4. Recombinant HER-2/neu(p185, ErbB-2(eBiosciences, Inc.)) was captured on the activated GLM sensorchip byinjecting 4.0 μg/mL in 10 mM NaOAc (pH 4.5) at 25 μL/min until approx.3000 resonance units (RUs) were immobilized with the remaining activegroups quenched. 40 μg/mL of purified anti-HER-2/neu antibodiescomprising the variant CH3 domains were indirectly captured on thesensorchip by binding the Her-2/neu protein when injected at 25 μL/minfor 240 s (resulting in approx. 500RUs) following a buffer injection toestablish a stable baseline. FcgammaR(CD16a(f allotype) and CD32b)concentrations (6000, 2000, 667, 222, and 74.0 nM) were injected at 60μL/min for 120 s with a 180 s dissociation phase to obtain a set ofbinding sensograms. Resultant K_(D) values were determined from bindingisotherms using the Equilibrium Fit model with reported values as themean of three independent runs. Comparisons were made with the wild-typeIgG1 Fc domain and binding is expressed as a ratio of the WT kD to thevariant kD (See, Table 5).

TABLE 5 Ratio of kD wild-type IgG1 to variant CH3 domain antibodybinding independently to CD16a and CD32b CD16a CD32b Ratio Ratio VariantWT/Variant WT/Variant Control 1 1.28 1.68 Control 2 1.1 1.13 AZ3 1.751.87 AZ6 1.38 1 AZ8 1.75 1.64 AZ12 N/A N/A AZ14 N/A N/A AZ15 0.72 0.59AZ16 0.95 0.64 AZ17 2.28 2.37 AZ18 1.53 1.7 AZ19 1.55 1.89 AZ20 2.561.93 AZ21 2.41 3.28 AZ22 2.02 2.37 AZ23 1 2.16 AZ24 1.79 2.26 AZ25 2.022.37 AZ26 2.38 2.59 AZ27 2.27 2.38 AZ28 1.45 2.15 AZ29 1.62 2.13 AZ301.61 2.38 AZ31 1.63 2.29 AZ32 1.82 2.48 AZ33 1.91 1.89 AZ34 1.88 1.88AZ38 1.78 1.44 AZ42 1.28 1.09 AZ43 1.63 1.73 AZ44 2.76 3.07 AZ46 2.162.66 AZ47 1.76 2.12 AZ48 2.02 1.59 AZ49 2.09 2.9 AZ50 2.33 1.86 AZ521.55 1.5 AZ53 1.87 1.27 AZ54 1.36 1.64 AZ58 2.33 1.48 AZ59 1.18 1.57AZ60 1.51 1.23 AZ61 1.41 1.75 AZ62 1.53 1.88 AZ63 0.9 0.95 AZ64 0.95 0.9AZ65 0.93 0.9 AZ66 1.26 1.19 AZ67 1.21 1.13 AZ68 1.02 1.1 AZ69 0.96 1.05AZ70 1.06 1.11 AZ71 0.89 0.95 AZ72 1.04 1.02 AZ73 1.09 1.07 AZ74 1.251.17 AZ75 1.34 1.22 AZ76 0.99 1 AZ77 1 1.08 AZ78 0.9 1 AZ79 1.01 0.8AZ81 1.01 0.84 AZ82 0.97 0.94 AZ83 0.94 0.94 AZ84 0.93 1 AZ85 1.01 1.14AZ86 1.22 1.18 AZ87 1.03 1.1 AZ88 1.11 1.15 AZ89 1.12 1.24 AZ91 1.111.11 AZ92 1.21 1.24 AZ93 1.21 1.18 AZ94 1.17 1.19 AZ95 0.86 0.96 AZ980.79 0.82 AZ99 1.16 1.15 AZ100 1.13 1.12 AZ101 1.24 1.23 AZ106 0.76 0.64AZ114 1.3 0.84 AZ115 1.13 0.82 AZ117 0.89 1 AZ122 0.89 0.92 AZ123 0.850.92 AZ124 0.99 1.09 AZ125 1 1 AZ126 0.86 0.9 AZ129 1.91 2.57 AZ130 1.912.54

Example 5 Rational Design of Fc Variants Using Fc_CH3Engineering—Scaffold 1 (1a and 1b) and the development of AZ17-62 andAZ133-AZ2438

To improve the initial negative design Fc variant AZ8 for stability andpurity, the structural and computational strategies described above wereemployed. (See, FIG. 24) For example, the in depth structure-functionanalysis of AZ8 provided a detailed understanding for each of theintroduced mutations of AZ8, L351Y_V397S_F405A_Y407V/K₃₉₂V_T394Wcompared to wild-type human IgG1 and indicated that the important coreheterodimer mutations were L351Y_F405A_Y407V/T394W, while V397S, K₃₉₂Vwere not relevant for heterodimer formation. The core mutations(L351Y_F405A_Y407V/T394W) are herein referred to as “Scaffold 1”mutations. The analysis furthermore revealed that the importantinterface hotspots that are lost with respect to wild-type (WT)homodimer formation are the interactions of WT-F405-K₄₀₉, Y407-T366 andthe packing of Y407-Y407 and -F405 (See, FIG. 29). This was reflected inthe packing, cavity and MD analysis, which showed a large conformationaldifference in the loop region D399-S400-D401 (See, FIG. 30) and theassociated β-sheets at K₃₇₀. This resulted in the loss of the interchaininteractions K₄₀₉-D399 (See, FIG. 30) and weakening of the strong K₃₇₀hydrogen bond to E357 (K₃₇₀ is no longer in direct contact with S364 andE357, but is entirely solvent exposed). In the WT IgG1 CH3 domain theseregions tether the interface at the rim protects the core interactionsfrom bulk solvent competition and increases the dynamic occurrence offavorable hydrophobic van der Waals interactions. The consequence was alower buried surface area of AZ8 compared to WT and a higher solventaccessibility of the hydrophobic core. This indicated the most importantfactors for the lower stability of AZ8 compared to WT stability was a)the loss of the WT-F405-K₄₀₉ interaction and packing of F405, and b) theloss of the strong packing interaction of Y407-Y407 and Y407-T366. See,FIG. 29

Consequently, we identified the key residues/sequence motifs responsiblefor the low stability of AZ8 compared to WT. To improve the stabilityand heterodimer specificity of AZ8 the subsequent positive designengineering efforts were therefore specifically focused on stabilizingthe loop conformation of positions 399-401 in a more ‘closed’—WT likeconformation (See, FIG. 30) and compensating for the overall slightlydecreased(looser) packing of the hydrophobic core at positions T366 andL368 (See, FIG. 29).

To achieve this stabilization of the loop conformation of positions399-401 the described computational approach was used to evaluate ourdifferent targeted design ideas. Specifically, three differentindependent options for Fc variant AZ8 were analyzed to optimize theidentified key regions for improving stability. First, the cavity closeto position K₄₀₉ and F405A was evaluated for better hydrophobic packingto both protect the hydrophobic core and stabilize the loop conformationof 399-400 (See, FIG. 30). Those included, but were not limited toadditional point mutations at positions F405 and K₃₉₂. Second, optionsfor improving the electrostatic interactions of positions 399-409 wereevaluated, to stabilize the loop conformation of 399-400 and protect thehydrophobic core. This included, but was not limited to additional pointmutations at positions T411 and S400. Third, the cavity at the corepacking positions T366, T394W and L368 was evaluated to improve the corehydrophobic packing (See, FIG. 29). Those included, but were not limitedto additional point mutations at positions T366 and L368. The differentindependent positive design ideas were tested in-silico and thebest-ranked variants using the computational tools (AZ17-AZ62) werevalidated experimentally for expression and stability as described inExamples 1-4. See Table 4 for a list of Fc variants from this designphase with a melting temperature of 70° C. or greater.

Fc variant AZ33 is an example of the development of an Fe variantwherein Scaffold 1 was modified resulting in Scaffold 1a mutations toimprove stability and purity. This Fc variant was designed based on AZ8with the goal improving the hydrophobic packing at positions 392-394-409and 366 to both protect the hydrophobic core and stabilize the loopconformation of 399-400. This Fc variant AZ33 heterodimer has twoadditional point mutations different from the core mutations of AZ8,K₃₉₂M and T366I. The mutations T366I_K₃₉₂M_T394W/F405A_Y407V are hereinreferred to as “Scaffold 1a” mutations. The mutation K₃₉₂M was designedto improve the packing at the cavity close to position K₄₀₉ and F405A toprotect the hydrophobic core and stabilize the loop conformation of399-400 (See, FIG. 31). T366I was designed to improve the corehydrophobic packing and to eliminate the formation of homodimers of theT394W chain (See, FIG. 29). The experimental data for AZ33 showedsignificantly improved stability over the initial negative design Fcvariant AZ8 (Tm 68° C.) wherein AZ33 has a Tm of 74° C. and aheterodimer content of >98%. (See, FIG. 25C)

Development of Fc Variants Using Scaffold 1 Mutations in Phase ThreeDesign of Fc Variant Heterodimers

Although AZ33 provides a significant stability and specificity (orpurity) improvement over the initial starting variant AZ8, our analysisindicates that further improvements to the stability of the Fc variantheterodimer can be made with further amino acid modifications using theexperimental data of AZ33 and the above described design methods. Thedifferent design ideas have been independently tested for expression andstability, but the independent design ideas are transferable and themost successful heterodimer will contain a combination of the differentdesigns. Specifically, for the optimization of AZ8 packing mutations atthe cavity close to K409-F405A-K392 have been evaluated independentlyfrom mutations that optimize the core packing at residues L366T-L368.These two regions 366-368 and 409-405-392 are distal from each other andare considered independent. Fc variant AZ33 for example has beenoptimized for packing at 409-405-392, but not at 366-368, because theseoptimization mutations were separately evaluated. The comparison of the366-368 mutations suggests that T366L has an improved stability overT366 and also T366I, the point mutation used in the development of Fcvariant AZ33. Consequently, the presented experimental data immediatelysuggest further optimization of AZ33 by introducing T366L instead ofT366I, for example. Therefore, the amino acid mutations in the CH3domain T366L_K₃₉₂M_T394W/F405A_Y407V are herein referred to as “Scaffold1b” mutations.

In a similar manner the complete experimental data has been analyzed toidentify point mutations that can be used to further improve the currentFc variant heterodimer AZ33. These identified mutations were analyzed bythe above described computational approach and ranked to yield the listof additional Fc variant heterodimers based on AZ33 as shown in Table 6.

Example 6 Rational Design of Fc Variants Using Fc_CH3Engineering—Scaffold 2 (a and b) and, the Development of AZ63-101 andAZ2199-AZ2524

To improve the initial negative design phase Fc variant AZ15 forstability and purity, the structural and computational strategiesdescribed above were employed (See, FIG. 24). For example, the in depthstructure-function analysis of Fc variant AZ15 provided a detailedunderstanding for each of the introduced mutations of AZ15,L351Y_Y407A/E357L_T366A_K409F_T411N compared to wild-type (WT) humanIgG1 and indicated that the important core heterodimer mutations wereL351Y_Y407A/T366A_K409F, while E357L, T411N were not directly relevantfor heterodimer formation and stability. The core mutations(L351Y_Y407A/T366A_K409F) are herein referred to as “Scaffold 2”mutations. The analysis furthermore revealed that the importantinterface hotspots that are lost with respect to wild-type (WT)homodimer formation are the salt bridge D399-K409, the hydrogen bondY407-T366 and the packing of Y407-Y407. Our detailed analysis, providedbelow, describes how we improved the stability of our original Fcvariant AZ15 and the positions and amino acid modifications made toachieve these Fc variants with improved stability.

Development of Fc Variants Using Scaffold 2 Mutations and the FurtherDevelopment of Scaffold 2a Mutations.

Our in-silico analysis indicated a non-optimal packing of the Fc variantAZ15 mutations K409F_T366A_Y407A and an overall decreased packing of thehydrophobic core due to the loss of the WT-Y407-Y407 interactions. Thepositive design efforts in the subsequent engineering phase were focusedon point mutations to compensate for these packing deficits in theinitial Fc variant AZ15. The targeted residues included positions T366,L351, and Y407. Different combinations of these were tested in-silicoand the best-ranked Fc variants using the computational tools(AZ63-AZ70) were validated experimentally for expression and stabilityas described in Examples 1-4.

Fc variant AZ70 is an example of the development of an Fc variantwherein Scaffold 2 was modified resulting in Scaffold 2a mutations toimprove stability and purity. This Fc variant was designed based on AZ15with the goal of achieving better packing at the hydrophobic core asdescribed above. Fc variant AZ70 has the same Scaffold 2 core mutations(L351Y_Y407A/T366A_K409F) as described above except that T366 wasmutated to T366V instead of T366A (FIG. 33). The L351Y mutation improvesthe 366A_409F/407A variant melting temperature from 71.5° C. to 74° C.,and the additional change from 366A to 366V improves the Tm to 75.5° C.(See, AZ63, AZ64 and AZ70 in Table 4, with a Tm of 71.5° C., 74° C. and75.5° C., respectively) The core mutations (L351Y_Y407A/T366V_K409F) areherein referred to as “Scaffold 2a” mutations. The experimental data forFc variant AZ70 showed significantly improved stability over the initialnegative design Fc variant AZ15 (Tm 71° C.) wherein AZ70 has a Tm of75.5° C. and a heterodimer content of >90% (FIGS. 33 and 27).

Development of Fc Variants Using Scaffold 2 Mutations and the FurtherDevelopment of Scaffold 2b Mutations.

The Molecular Dynamics simulation (MD) and packing analysis showed apreferred more ‘open’ conformation of the loop 399-400, which was likelydue to the loss of the WT salt bridge K409-D399. This also results inthe unsatisfied D399, which in turn preferred a compensating interactionwith K392 and induced a more ‘open’ conformation of the loop. This more‘open’ loop conformation results in an overall decreased packing andhigher solvent accessibility of the core CH3 domain interface residues,which in turn significantly destabilized the heterodimer complex.Therefore, one of the targeted positive design efforts was the tetheringof this loop in a more ‘closed’, WT-like conformation by additionalpoint mutations that compensate for the loss of the D399-K409 saltbridge and the packing interactions of K409. The targeted residuesincluded positions T411, D399, S400, F405, N390, K392 and combinationsthereof. Different packing, hydrophobic- and electrostatic positiveengineering strategies were tested in silico with respect to the abovepositions and the best-ranked Fc variants determined using thecomputational tools (AZ71-AZ101) were validated experimentally forexpression and stability as described in Examples 1-4.

Fc variant AZ94 is an example of the development of an Fc variantwherein Scaffold 2 is modified resulting in Scaffold 2b mutations alongwith additional point mutations to improve stability and purity. This Fcvariant was designed based on AZ15 with the goal of tethering loop399-400 in a more ‘closed’, WT-like conformation and compensating forthe loss of the D399-K409 salt bridge as described above. Fc variantAZ94 has four additional point mutations to Scaffold 2(L351Y_Y407A/T366A_K409F) and returns L351Y to wild-type L351 leaving.(Y407A/T366A_K409F) as the core mutations for this Fc variant. The coremutations Y407A/T366A_K409F are herein referred to as “Scaffold 2b”mutations. The four additional point mutations of AZ94 areK392E_T411E/D399R_S400R. The mutations T411E/D399R were engineered toform an additional salt bridge and compensate for the loss of theK409/D399 interaction (FIG. 34). Additionally, this salt bridge wasdesigned to prevent homodimer formation by disfavoring charge-chargeinteractions in both potential homodimers. The additional mutationsK392E/S400R were intended to form another salt bridge and hence furthertether the 399_400 loop in a more ‘closed’, WT-like conformation (FIG.34). The experimental data for AZ94 showed improved stability and purityover the initial negative design Fc variant AZ15 (Tm 71° C., >90%purity) wherein Fc variant AZ94 has a Tm of 74° C. and a heterodimercontent or purity of >95%.

Development of Fc Variants Using Scaffold 2 Mutations in Phase ThreeDesign of Fc Variant Heterodimers

Both Fc variants AZ70 and AZ94 provide a significant improvement instability and purity over the initial negative design Fc variant AZ15,but our analysis and the comparison of AZ70 and AZ94 directly indicatethat further improvements to the stability of the Fc variant heterodimercan be made with further amino acid modifications. For example, Fcvariants AZ70 and AZ94 were designed to target two distinctnon-optimized regions in the initial variant AZ15, which wasaccomplished by improving the packing at the hydrophobic core and makingmutations outside of the core interface residues resulting in additionalsalt bridges and hydrogen bonding to stabilize the loop conformation ofpositions 399-401. The additional point mutations of Fc variants AZ70and AZ94 are distal from each other and are therefore independent andtransferable to other Fc variants designed around the same Scaffold 2core mutations, including 2a and 2b mutations. Specifically, AZ70 onlycarries the optimized core mutations L351Y_Y407A/T366A_K409F, but noadditional salt bridges, whereas AZ94 comprises four additionalelectrostatic mutations (K392E_T411E/D399R_S400R), but has one lessmutation in the hydrophobic core interface (Y407A/T366A_K409F). TheseScaffold 2b mutations are less stable than AZ70 (See, for example AZ63,which has equivalent core mutations as AZ94 and Tm of 72° C.), but arecompensated for by the addition of K392E_T411E/D399R_S400R mutations.The presented experimental stability and purity data indicates thatcombining the mutations of AZ70, which optimizes the hydrophobic core,and the electrostatic mutations of AZ94 should further improve stabilityand purity of the Fc variant heterodimers. In a similar manner thecomplete experimental data for Scaffold 2 Fc variants (AZ63-101) hasbeen analyzed to identify point mutations that can be used to furtherimprove the Fc variant heterodimers AZ70 and AZ94. These identifiedmutations were further analyzed by the above described computationalapproach and ranked to yield the list of additional Fc variantheterodimers based on AZ70 and AZ94 as shown in Table 7.

Example 7 Effect of Heterodimeric CH3 on FcgR Binding

As a prototypical example of heterodimeric Fc activity with FcgR, wehave tested two variant antibodies with heterodimeric Fc regionA:K409D_K392D/B:D399K_D356K (Control 1 (het 1 in FIG. 35)) andA:Y349C_T366S_L368A_Y407V/B:S354C_T366W (Control 4 (het 2 in FIG. 35))with Her2 binding Fab arms in an SPR assay described in Example 4 forFcgR binding. As shown in FIG. 35, we observe that both theheterodimeric Fc regions bind the different Fcgamma receptors with thesame relative strength as the wild type IgG1 Fc region, but overall, theheterodimeric Fc region bound each of the FcgR's slightly better thanthe wild type antibody. This indicates that mutations at the CH3interface of Fc can impact the binding strength of the Fc region forFcgamma receptors across the CH2 domains as observed in our moleculardynamics simulations and analysis.

Example 8 Effect of Asymetric Mutations in CH₂ of a Heterodimeric Fc onFcgR Binding

Mutation of Serine at position 267 in the CH2 domain of the Fc region toan Aspartic acid (S267D) is known to enhance binding to Fcgamma IIbF,IIbY & IIaR receptors when introduced in a homodimeric manner in the twochains of CH2 domain. This mutation can be introduced on only one of theCH2 domains in an heterodimeric Fc molecule to gain roughly half theimprovement in binding strength relative to when this mutation isintroduced in a homodimeric CH2 Fc as the data presented in FIG. 36Aindicates. On the other hand, the E269K mutation in a homodimeric CH2domain of Fc prevents binding of the Fc region to FcgR. We present ascheme for enhanced manipulation of the binding strength of the Fcregion for the Fcg Receptors by the asymmetric introduction of thesefavorable and unfavorable mutations on one of the two chains in the CH2domain of the Fe. The introduction of E269K mutation in an asymmetricmanner on one CH2 chain in a heterodimeric Fc acts as a polarity driverby blocking binding of the FcgR at the face where it is present, whileletting the other face of the Fc interact with the FcgR in a normalmanner. The results from this experimentation are presented in FIG. 36A.The opportunity to selectively alter the binding strength via both thechains of Fc in an independent manner provides increased opportunity tomanipulate the binding strength and selectivity between Fc and FcgReceptors. Thus, such asymmetric design of mutations in the CH2 domainallows us to introduce positive and negative design strategies to favoror disfavor certain binding models, providing greater opportunity tointroduce selectivity.

In a subsequent experiment, we have altered the selectivity profile ofthe base Fc mutant S239D_D265S_I332E_S298A that shows increased bindingstrength to the Fcgamma IIIaF and IIIaV receptors while continuing toexhibit weaker binding to the Fcgamma IIaR, IIbF and IIbY receptors.This is shown in the binding profile shown in FIG. 36B. By introducingasymmetric mutations E269K in chain A and avoiding the I332E mutation inchain B, we are able to generate a novel FcgR binding profile thatfurther weakens IIa and IIb receptor binding and makes the Fc morespecific for the IIIa receptor binding.

In another example shown in FIG. 36C, asymmetric mutations arehighlighted relative to the homodimeric Fc involving the mutationS239D/K326E/A330L/I332E/S298A in the CH2 domain. Relative to the wildtype IgG1 Fc, this variant show increased binding to the 111a receptorbut also binds the IIa and IIb receptors slightly stronger than the wildtype Fc. Introduction of these mutations in an asymmetric mannerA:S239D/K326E/A330L/I332E and B:S298A while reducing the IIIa binding,also increases the IIa/IIb receptor binding, loosing selectivity in theprocess. By introducing an asymmetirc E269K mutation in thisheterodimeric variant, i.e. A:S239D/K326E/A330L/I332E/E269K and B:S298A,we are able to reduce the IIa/IIb binding back to wild type levels. Thishighlights the fact that the use of asymmetric mutations in the CH2domain of Fc is able to provide significant opportunity to designimproved FcgammaR selectivity.

The reagents employed in the examples are commercially available or canbe prepared using commercially available instrumentation, methods, orreagents known in the art. The foregoing examples illustrate variousaspects of the invention and practice of the methods of the invention.The examples are not intended to provide an exhaustive description ofthe many different embodiments of the invention. Thus, although theforgoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, thoseof ordinary skill in the art will realize readily that many changes andmodifications can be made thereto without departing from the spirit orscope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

We claim:
 1. An isolated heteromultimer comprising a heterodimeric CH3domain comprising a first CH3 domain polypeptide and a second CH3 domainpolypeptide, the first CH3 domain polypeptide comprising amino acidmodifications at positions F405 and Y407, and the second CH3 domainpolypeptide comprising amino acid modifications at positions T366 andT394, wherein: (i) the first CH3 domain polypeptide further comprises anamino acid modification at position L351, and (ii) the second CH3 domainpolypeptide further comprises an amino acid modification at positionK392, wherein the amino acid modification at position F405 is F405A,F405T, F405S or F405V; and the amino acid modification at position Y407is Y407V, Y407A, Y407L or Y407I; the amino acid modification at positionT394 is T394W; the amino acid modification at position L351 is L351Y;the amino acid modification at position K392 is K392L, K392M, K392V orK392F, and the amino acid modification at position T366 is T366I, T366L,T366M or T366V, wherein the heterodimeric CH3 domain has a meltingtemperature (Tm) of about 70° C. or greater and a purity greater thanabout 90%, and wherein the numbering of amino acid residues is accordingto the EU index as set forth in Kabat.
 2. The isolated heteromultimeraccording to claim 1, wherein the amino acid modification at positionF405 is F405A.
 3. The isolated heteromultimer according to claim 1,wherein the amino acid modification at position T366 is T366I or T366L.4. The isolated heteromultimer to claim 1, wherein the amino acidmodification at position Y407 is Y407V.
 5. The isolated heteromultimeraccording to claim 1, wherein the amino acid modification at positionF405 is F405A, the amino acid modification at position Y407 is Y407V,the amino acid modification at position T366 is T366I or T366L, and theamino acid modification at position K392 is K392L or K392M.
 6. Theisolated heteromultimer according to claim 1, wherein the amino acidmodification at position F405 is F405A, the amino acid modification atposition Y407 is Y407V, the amino acid modification at position T366 isT366L, and the amino acid modification at position K392 is K392M.
 7. Theisolated heteromultimer according to claim 1, wherein the amino acidmodification at position F405 is F405A, the amino acid modification atposition Y407 is Y407V, the amino acid modification at position T366 isT366L, and the amino acid modification at position K392 is K392L.
 8. Theisolated heteromultimer according to claim 1, wherein the amino acidmodification at position F405 is F405A, the amino acid modification atposition Y407 is Y407V, the amino acid modification at position T366 isT366I, and the amino acid modification at position K392 is K392M.
 9. Theisolated heteromultimer according to claim 1, wherein the amino acidmodification at position F405 is F405A, the amino acid modification atposition Y407 is Y407V, the amino acid modification at position T366 isT366I, and the amino acid modification at position K392 is K392L. 10.The isolated heteromultimer according to claim 1, wherein theheterodimeric CH3 domain is comprised by an Fc region based on an IgG Fcregion.
 11. The isolated heteromultimer according to claim 10, whereinthe IgG Fc region is an IgG1 Fc region.
 12. The isolated heteromultimeraccording to claim 1, wherein the heteromultimer is a bispecific ormultispecific antibody.
 13. The isolated heteromultimer according toclaim 12, wherein the bispecific or multispecific antibody binds atleast one cancer antigen.
 14. The isolated heteromultimer according toclaim 13, wherein the bispecific or multispecific antibody is conjugatedto a therapeutic agent.
 15. The isolated heteromultimer according toclaim 1, wherein the first CH3 domain polypeptide further comprises anamino acid modification at position S400 selected from S400D and S400E.16. The isolated heteromultimer according to claim 1, wherein the secondCH3 domain polypeptide further comprises the amino acid modificationN390R.
 17. The isolated heteromultimer according to claim 1, wherein thefirst CH3 domain polypeptide further comprises an amino acidmodification at position S400 selected from S400D and S400E, and thesecond CH3 domain polypeptide further comprises the amino acidmodification N390R.
 18. The isolated heteromultimer according to claim17, wherein the amino acid modification at position F405 is F405A, theamino acid modification at position Y407 is Y405V, the amino acidmodification at position S400 is S400E, the amino acid modification atposition T366 is T366L, and the amino acid modification at position K392is K392M.
 19. An isolated heteromultimer comprising a heterodimeric CH3domain comprising: a first CH3 domain polypeptide comprising amino acidmodifications L351Y, F405A and Y407V, and a second CH3 domainpolypeptide comprising amino acid modifications T366L, K392M and T394W.20. An isolated heteromultimer comprising a heterodimeric CH3 domaincomprising: a first CH3 domain polypeptide comprising amino acidmodifications L351Y, F405A and Y407V, and a second CH3 domainpolypeptide comprising amino acid modifications T366L, K392L and T394W.21. An isolated heteromultimer comprising a heterodimeric CH3 domaincomprising: a first CH3 domain polypeptide comprising amino acidmodifications L351Y, S400E, F405A and Y407V, and a second CH3 domainpolypeptide comprising amino acid modifications T366L, N390R, K392M andT394W.
 22. An isolated heteromultimer comprising a heterodimeric CH3domain comprising: a first CH3 domain polypeptide comprising amino acidmodifications L351Y, S400E, F405A and Y407V, and a second CH3 domainpolypeptide comprising amino acid modifications T366L, N390R, K392L andT394W.
 23. The isolated heteromultimer according to claim 19, whereinthe heterodimeric CH3 domain is comprised by an Fc region based on anIgG1 Fc region.
 24. The isolated heteromultimer according to claim 23,wherein the heteromultimer is a bispecific antibody.
 25. The isolatedheteromultimer according to claim 20, wherein the heterodimeric CH3domain is comprised by an Fc region based on an IgG1 Fc region.
 26. Theisolated heteromultimer according to claim 25, wherein theheteromultimer is a bispecific antibody.
 27. The isolated heteromultimeraccording to claim 21, wherein the heterodimeric CH3 domain is comprisedby an Fc region based on an IgG1 Fc region.
 28. The isolatedheteromultimer according to claim 27, wherein the heteromultimer is abispecific antibody.
 29. The isolated heteromultimer according to claim22, wherein the heterodimeric CH3 domain is comprised by an Fc regionbased on an IgG1 Fc region.
 30. The isolated heteromultimer according toclaim 29, wherein the heteromultimer is a bispecific antibody.